Subunits of NADH dehydrogenase

ABSTRACT

The present invention provides four NADH dehydrogenase subunits (designated individually as NDS-1, NDS-2, NDS-3, and NDS-4 and collectively as NDS) and polynucleotides which identify and encode NDS. The invention also provides genetically engineered expression vectors and host cells comprising the nucleic acid sequences encoding NDS and a method for producing NDS. The invention also provides for use of NDS and agonists, antibodies, or antagonists specifically binding NDS, in the prevention and treatment of diseases associated with expression of NDS. Additionally, the invention provides for the use of antisense molecules to polynucleotides encoding NDS for the treatment of diseases associated with the expression of NDS. The invention also provides diagnostic assays which utilize the polynucleotide, or fragments or the complement thereof, and antibodies specifically binding NDS.

This application is a divisional application of U.S. application Ser.No. 08/785,065, filed Jan. 17, 1997 now U.S. Pat. No. 5,814,451.

FIELD OF THE INVENTION

This invention relates to nucleic acid and amino acid sequences ofpolypeptide subunits of NADH dehydrogenase and to the use of thesesequences in the diagnosis, prevention, and treatment of cancer,myopathies, neurodegenerative diseases, immune system disorders, anddiseases and disorders of the sympathetic nervous system.

BACKGROUND OF THE INVENTION

NADH dehydrogenase (NADH:ubiquinone oxidoreductase, NADH-D) is the firstmultienzyme complex (Complex I) in a chain of three complexes that makeup the mitochondrial electron transport chain. The mitochondrialelectron transport chain is responsible for the transport of electronsfrom NADH to oxygen and the coupling of this oxidation to the synthesisof ATP (oxidative phosphorylation) which provides the energy source fordriving a cell's many energy-requiring reactions. NADH-D accomplishesthe first step in this process by accepting electrons from NADH andpassing them through a flavin molecule to ubiquinone, which transfersthe electrons to the second enzyme complex in the chain.

NADH-D and the other members of the electron transport chain are locatedin the mitochondrial membrane. NADH-D is the largest of the threecomplexes with an estimated mass of 800 kDa comprising some 40polypeptides of widely varying size and composition. The polypeptidecomposition of NADH-D in a variety of mammalian species including rat,rabbit, cow, and man is very similar (Cleeter, M. W. J. and Ragan, C. I.(1985) Biochem. J. 230: 739-46). The best characterized NADH-D is frombovine heart mitochondria and is composed of 41 polypeptide chains(Walker, J. E. et al. (1992) J. Mol. Biol. 226: 1051-72). Seven of thesepolypeptides are encoded by mitochondrial DNA while the remaining 34 arenuclear gene products that are imported into the mitochondria. Theseimported polypeptides are characterized by various N-terminal peptidesequences or modified N-terminal amino acids (myristoylation oracetylation) that target them to the mitochondria and are then cleavedfrom the mature protein. The 24-, 51-, and 75-kDa subunits have beenidentified as being catalytically important in electron transport, withthe 51-kDa subunit forming part of the NADH binding site and containingthe flavin moiety that is the initial electron acceptor. The location ofother functionally important groups, such as the electron-carrying ironsulfate centers, remains to be determined. Many of the smaller subunits(<30 kDa) may play a purely structural role in the complex.

Defects and altered expression of NADH-D are associated with a varietyof disease conditions in man, incluing; neurodegenerative diseases,myopathies, and cancer. In addition, NADH-D reduction of the quinonemoiety in chemotherapeutic agents such as doxorubicin is believed tocontribute to the antitumor activity and/or mutagenicity of these drugs.

The discovery of polynucleotides encoding NADH dehydrogenase, and themolecules themselves, provides a means to investigate the control ofcellular respiration under normal and disease conditions. Such moleculesrelated to NADH dehydrogenase satisfy a need in the art by providing newdiagnostic or therapeutic compositions useful in diagnosing and treatingcancers, myopathies, neurodegenerative diseases, and diseases anddisorders of the sympathetic nervous system.

SUMMARY OF THE INVENTION

The present invention features four NADH dehydrogenase subunits,designated individually as NDS-1, NDS-2, NDS-3 and NDS-4 andcollectively as NDS, and characterized as having similarity to NADHdehydrogenase.

Accordingly, the invention features substantially purified NDS proteinsNDS-1, NDS-2, NDS-3, and NDS-4 having the amino acid sequences shown inSEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO:7, respectively.

One aspect of the invention features isolated and substantially purifiedpolynucleotides that encode NDS proteins. In a particular aspect, thepolynucleotides are the nucleotide sequences of SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6, or SEQ ID NO:8 respectively.

The invention also features a polynucleotide sequence comprising thecomplement of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 orvariants thereof. In addition, the invention features polynucleotidesequences which hybridize under stringent conditions to SEQ ID NO:2, SEQID NO:4, SEQ ID NO:6, or SEQ ID NO:8.

The invention additionally features nucleic acid sequences encodingpolypeptides, oligonucleotides, peptide nucleic acids (PNA), fragments,portions or antisense molecules thereof, and expression vectors and hostcells comprising polynucleotides that encode NDS. The present inventionalso features antibodies which bind specifically to NDS, andpharmaceutical compositions comprising substantially purified NDS. Theinvention also features the use of agonists and antagonists of NDS.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B, and 1C show the amino acid sequence (SEQ ID NO:1 andnucleic acid sequence (SEQ ID NO:2) of NDS-1. The alignment was producedusing MacDNASIS PRO software (Hitachi Software Engineering Co., Ltd.,San Bruno, Calif.).

FIGS. 2A and 2B show the amino acid sequence (SEQ ID NO:3) and nucleicacid sequence (SEQ ID NO:4) of NDS-2.

FIGS. 3A and 3B show the amino acid sequence (SEQ ID NO:5) and nucleicacid sequence (SEQ ID NO:6) of NDS-3.

FIGS. 4A and 4B show the amino acid sequence (SEQ ID NO:7) and nucleicacid sequence (SEQ ID NO:8) of NDS-4.

FIG. 5 shows the amino acid sequence alignments between NDS-1 (SEQ IDNO:1) and the bovine heart 30-kDa subunit (GI 163416; SEQ ID NO:9). Thealignment was produced using the multisequence alignment program of(DNASTAR Inc., Madison, Wis.).

FIG. 6 shows the amino acid sequence alignments between NDS-2 (SEQ IDNO:3) and the bovine heart B 15 subunit (GI 114; SEQ ID NO:10).

FIG. 7 shows the amino acid sequence alignments between NDS-3 (SEQ IDNO:5) and the bovine heart 15-kDa (IP) subunit (GI 224; SEQ ID NO:11).

FIG. 8 shows the amino acid sequence alignments between NDS-4 (SEQ IDNO:7) and the bovine heart B14.5b subunit (GI 582; SEQ ID NO:12).

FIGS. 9A and 9B show the hydrophobicity plots (MACDNASIS PRO software)for NDS-1, (SEQ ID NO:1) and bovine heart 30-kDa subunit (SEQ ID NO:9).The positive X axis reflects amino acid position, and the negative Yaxis reflects hydrophobicity.

FIGS. 10A and 10B show the hydrophobicity plots for NDS-2, (SEQ ID NO:3)and bovine heart B15 subunit, (SEQ ID NO:10).

FIGS. 11A and 11B show the hydrophobicity plots for NDS-3, (SEQ ID NO:5)and bovine heart 15-kDa (IP) subunit, (SEQ ID NO:11).

FIGS. 12A and 12B show the hydrophobicity plots for NDS-4, (SEQ ID NO:7)and bovine heart B14.5b subunit, (SEQ ID NO:12).

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleotide sequences, and methods aredescribed, it is understood that this invention is not limited to theparticular methodology, protocols, cell lines, vectors, and reagentsdescribed as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “ahost cell” includes a plurality of such host cells, reference to the“antibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are now described. All publications mentionedherein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologieswhich are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure by virtueof prior invention.

Definitions

“Nucleic acid sequence”, as used herein, refers to an oligonucleotide,nucleotide, or polynucleotide, and fragments or portions thereof, and toDNA or RNA of genomic or synthetic origin which may be single- ordouble-stranded and represent the sense or antisense strand. Similarly,“amino acid sequence” as used herein refers to an oligopeptide, peptide,polypeptide, or protein sequence, and fragments or portions thereof, andto naturally occurring or synthetic molecules.

Where “amino acid sequence” is recited herein to refer to an amino acidsequence of a naturally occurring protein molecule, “amino acidsequence” and like terms, such as “polypeptide” or “protein” are notmeant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule.

“Peptide nucleic acid”, as used herein, refers to a molecule whichcomprises an oligomer to which an amino acid residue, such as lysine,and an amino group have been added. These small molecules, alsodesignated anti-gene agents, stop transcript elongation by binding totheir complementary strand of nucleic acid (Nielsen, P. E. et al. (1993)Anticancer Drug Des. 8:53-63).

NDS, as used herein, refers to the amino acid sequences of substantiallypurified NDS obtained from any species, particularly mammalian,including bovine, ovine, porcine, murine, equine, and preferably human,from any source whether natural, synthetic, semi-synthetic, orrecombinant.

“Consensus”, as used herein, refers to a nucleic acid sequence which hasbeen resequenced to resolve uncalled bases, or which has been extendedusing XL-PCR (Perkin Elmer, Norwalk, Conn.) in the 5′ and/or the 3′direction and resequenced, or which has been assembled from theoverlapping sequences of more than one Incyte clone using the GELVIEWfragment assembly system (GCG, Madison, Wis.), or which has been bothextended and assembled.

A “variant” of NDS, as used herein, refers to an amino acid sequencethat is altered by one or more amino acids. The variant may have“conservative” changes, wherein a substituted amino acid has similarstructural or chemical properties, e.g., replacement of leucine withisoleucine. More rarely, a variant may have “nonconservative” changes,e.g., replacement of a glycine with a tryptophan. Similar minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, DNASTAR software.

A “deletion”, as used herein, refers to a change in either amino acid ornucleotide sequence in which one or more amino acid or nucleotideresidues, respectively, are absent.

An “insertion” or “addition”, as used herein, refers to a change in anamino acid or nucleotide sequence resulting in the addition of one ormore amino acid or nucleotide residues, respectively, as compared to thenaturally occurring molecule.

A “substitution”, as used herein, refers to the replacement of one ormore amino acids or nucleotides by different amino acids or nucleotides,respectively.

The term “biologically active”, as used herein, refers to a proteinhaving structural, regulatory, or biochemical functions of a naturallyoccurring molecule. Likewise, “immunologically active” refers to thecapability of the natural, recombinant, or synthetic NDS, or anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

The term “agonist”, as used herein, refers to a molecule which, whenbound to NDS, causes a change in NDS which modulates the activity ofNDS. Agonists may include proteins, nucleic acids, carbohydrates, or anyother molecules which bind to NDS.

The terms “antagonist” or “inhibitor”, as used herein, refer to amolecule which, when bound to NDS, blocks or modulates the biological orimmunological activity of NDS. Antagonists and inhibitors may includeproteins, nucleic acids, carbohydrates, or any other molecules whichbind to NDS.

The term “modulate”, as used herein, refers to a change or an alterationin the biological activity of NDS. Modulation may be an increase or adecrease in protein activity, a change in binding characteristics, orany other change in the biological, functional or immunologicalproperties of NDS.

The term “mimetic”, as used herein, refers to a molecule, the structureof which is developed from knowledge of the structure of NDS or portionsthereof and, as such, is able to effect some or all of the actions ofNADH-D protein-like molecules.

The term “derivative”, as used herein, refers to the chemicalmodification of a nucleic acid encoding NDS or the encoded NDS.Illustrative of such modifications would be replacement of hydrogen byan alkyl, acyl, or amino group. A nucleic acid derivative would encode apolypeptide which retains essential biological characteristics of thenatural molecule.

The term “substantially purified”, as used herein, refers to nucleic oramino acid sequences that are removed from their natural environment,isolated or separated, and are at least 60% free, preferably 75% free,and most preferably 90% free from other components with which they arenaturally associated.

“Amplification”, as used herein, refers to the production of additionalcopies of a nucleic acid sequence and is generally carried out usingpolymerase chain reaction (PCR) technologies well known in the art(Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, a LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y.).

The term “hybridization”, as used herein, refers to any process by whicha strand of nucleic acid binds with a complementary strand through basepairing.

The term “hybridization complex”, as used herein, refers to a complexformed between two nucleic acid sequences by virtue of the formation ofhydrogen binds between complementary G and C bases and betweencomplementary A and T bases; these hydrogen bonds may be firtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., C₀t or R₀tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,membranes, filters, chips, pins or glass slides to which cells have beenfixed for in situ hybridization).

The terms “complementary” or “complementarity”, as used herein, refer tothe natural binding of polynucleotides under permissive salt andtemperature conditions by base-pairing. For example, for the sequence“A-G-T” binds to the complementary sequence “T-C-A”. Complementaritybetween two single-stranded molecules may be “partial”, in which onlysome of the nucleic acids bind, or it may be complete when totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions,which depend upon binding between nucleic acids strands.

The term “homology”, as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence is one that atleast partially inhibits an identical sequence from hybridizing to atarget nucleic acid; it is referred to using the functional term“substantially homologous.” The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or northern blot, solutionhybridization and the like) under conditions of low stringency. Asubstantially homologous sequence or probe will compete for and inhibitthe binding (i.e., the hybridization) of a completely homologoussequence or probe to the target sequence under conditions of lowstringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction. The absence of non-specific binding maybe tested by the use of a second target sequence which lacks even apartial degree of complementarity (e.g., less than about 30% identity);in the absence of non-specific binding, the probe will not hybridize tothe second non-complementary target sequence.

As known in the art, numerous equivalent conditions may be employed tocomprise either low or high stringency conditions. Factors such as thelength and nature (DNA, RNA, base composition) of the sequence, natureof the target (DNA, RNA, base composition, presence in solution orimmobilization, etc.), and the concentration of the salts and othercomponents (e.g., the presence or absence of formamide, dextran sulfateand/or polyethylene glycol) are considered and the hybridizationsolution may be varied to generate conditions of either low or highstringency different from, but equivalent to, the above listedconditions.

The term “stringent conditions”, as used herein, is the “stringency”which occurs within a range from about Tm-5° C. (5° C. below the meltingtemperature (Tm) of the probe) to about 20° C. to 25° C. below Tm. Aswill be understood by those of skill in the art, the stringency ofhybridization may be altered in order to identify or detect identical orrelated polynucleotide sequences.

The term “antisense”, as used herein, refers to nucleotide sequenceswhich are complementary to a specific DNA or RNA sequence. The term“antisense strand” is used in reference to a nucleic acid strand that iscomplementary to the “sense” strand. Antisense molecules may be producedby any method, including synthesis by ligating the gene(s) of interestin a reverse orientation to a viral promoter which permits the synthesisof a complementary strand. Once introduced into a cell, this transcribedstrand combines with natural sequences produced by the cell to formduplexes. These duplexes then block either the further transcription ortranslation. In this manner, mutant phenotypes may be generated. Thedesignation “negative” is sometimes used in reference to the antisensestrand, and “positive” is sometimes used in reference to the sensestrand.

The term “portion”, as used herein, with regard to a protein (as in “aportion of a given protein”) refers to fragments of that protein. Thefragments may range in size from four amino acid residues to the entireamino acid sequence minus one amino acid. Thus, a protein “comprising atleast a portion of the amino acid sequence of SEQ ID NO:1” encompassesthe full-length human NDS and fragments thereof.

“Transformation”, as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method is selected based on the host cell being transformedand may include, but is not limited to, viral infection,electroporation, lipofection, and particle bombardment. Such“transformed” cells include stably transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as part of the host chromosome. They also includecells which transiently express the inserted DNA or RNA for limitedperiods of time.

The term “antigenic determinant”, as used herein, refers to that portionof a molecule that makes contact with a particular antibody (i.e., anepitope). When a protein or fragment of a protein is used to immunize ahost animal, numerous regions of the protein may induce the productionof antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

The terms “specific binding” or “specifically binding”, as used herein,in reference to the interaction of an antibody and a protein or peptide,mean that the interaction is dependent upon the presence of a particularstructure (i.e., the antigenic determinant or epitope) on the protein;in other words, the antibody is recognizing and binding to a specificprotein structure rather than to proteins in general. For example, if anantibody is specific for epitope “A”, the presence of a proteincontaining epitope A (or free, unlabeled A) in a reaction containinglabeled “A” and the antibody will reduce the amount of labeled A boundto the antibody.

The term “sample”, as used herein, is used in its broadest sense. Abiological sample suspected of containing nucleic acid encoding NDS orfragments thereof may comprise a cell, chromosomes isolated from a cell(e.g., a spread of metaphase chromosomes), genomic DNA (in solution orbound to a solid support such as for Southern analysis), RNA (insolution or bound to a solid support such as for northern analysis),cDNA (in solution or bound to a solid support), an extract from cells ora tissue, and the like.

The term “correlates with expression of a polynucleotide”, as usedherein, indicates that the detection of the presence of ribonucleic acidthat is similar to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8by northern analysis is indicative of the presence of mRNA encoding NDSin a sample and thereby correlates with expression of the transcriptfrom the polynucleotide encoding the protein.

“Alterations” in the polynucleotide of SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:6, or SEQ ID NO:8 as used herein, comprise any alteration in thesequence of polynucleotides encoding NDS including deletions,insertions, and point mutations that may be detected using hybridizationassays. Included within this definition is the detection of alterationsto the genomic DNA sequence which encodes NDS (e.g., by alterations inthe pattern of restriction fragment length polymorphisms capable ofhybridizing to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8),the inability of a selected fragment of SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:6, or SEQ ID NO:8 to hybridize to a sample of genomic DNA (e.g.,using allele-specific oligonucleotide probes), and improper orunexpected hybridization, such as hybridization to a locus other thanthe normal chromosomal locus for the polynucleotide sequence encodingNDS (e.g., using fluorescent in situ hybridization (FISH) to metaphasechromosomes spreads).

As used herein, the term “antibody” refers to intact molecules as wellas fragments thereof, such as Fab, F(ab′)₂, and Fv, which are capable ofbinding the epitopic determinant. Antibodies that bind NDS polypeptidescan be prepared using intact polypeptides or fragments containing smallpeptides of interest as the immunizing antigen. The polypeptide orpeptide used to immunize an animal can be derived from the translationof mRNA or synthesized chemically, and can be conjugated to a carrierprotein, if desired. Commonly used carriers that are chemically coupledto peptides include bovine serum albumin and thyroglobulin. The coupledpeptide is then used to immunize the animal (e.g., a mouse, a rat, or arabbit).

The term “humanized antibody”, as used herein, refers to antibodymolecules in which amino acids have been replaced in the non-antigenbinding regions in order to more closely resemble a human antibody,while still retaining the original binding ability.

The Invention

The invention is based on the discovery of NADH dehydrogenase subunits(NDS-1, NDS-2, NDS-3, and NDS-4, collectively referred to as NDS), thepolynucleotides encoding NDS, and the use of these compositions for thediagnosis, prevention, or treatment of cancers, myopathies,neurodegenerative diseases, and diseases and disorders of thesympathetic nervous system.

Nucleic acid sequences encoding the human NDS-1 of the present inventionwere first identified in Incyte Clone 4401 from a human mast cell line,HMC-1cDNA library (HMC1NOT01) through a computer-generated search foramino acid sequence alignments. A consensus sequence, SEQ ID NO:2, wasderived from this same clone.

Nucleic acid sequences encoding the human NDS-2 of the present inventionwere first identified in Incyte Clone 1600202 from a bladder tissue cDNAlibrary (BLADNOT03) through a computer-generated search for amino acidsequence alignments. A consensus sequence, SEQ ID NO:4, was derived fromthe following overlapping and/or extended nucleic acid sequences (cDNAlibrary from which derived): Incyte Clones 571087 (MMLR3DT01); 966781(BRSTNOT05); 1600202 (BLADNOT03); and 1913211 (PROSTUT04).

Nucleic acid sequences encoding the human NDS-3 of the present inventionwere first identified in Incyte Clone 1708121, from a prostate tissuecDNA library (PROSNOT16) through a computer-generated search for aminoacid sequence alignments. A consensus sequence, SEQ ID NO:6, was derivedfrom the following overlapping and/or extended nucleic acid sequences(cDNA library from which derived): Incyte Clones 928553 (BRAINOT04);957042 (KIDNNOT05); and 1708121 (PROSNOT16).

Nucleic acid sequences encoding the human NDS-4 of the present inventionwere first identified in Incyte Clone 1996788, from a breast tumortissue cDNA library (BRSTTUT03) through a computer-generated search foramino acid sequence alignments. A consensus sequence, SEQ ID NO:6, wasderived from the following overlapping and/or extended nucleic acidsequences (cDNA library from which derived): Incyte Clones 623462(PGANNOT01); 835412 (PROSNOT07); 1381432 (BRAITUT08); and 1996788(BRSTTUT03).

In one embodiment, the invention encompasses a polypeptide comprisingthe amino acid sequence of SEQ ID NO:1, and shown in FIGS. 1A, 1B, and1C. As shown in FIG. 5, NDS-1 has chemical and structural homology withbovine 30-kDa subunit (GI 163416). In particular, NDS-1 shares 90%overall identity with bovine 30-kDa subunit. An N-terminal signalsequence in the bovine 30-kDa subunit that extends from residues M1 toR37 and serves to direct the protein to the mitochondria is wellconserved in NDS-1. The sequence is cleaved in the mature proteinfollowing the translocation process. In particular, a high content ofarginine and alanine that is characteristic of these signal sequences isapparent in both proteins, as is the presence of an arginine at position−2 relative to the cleavage point at E37. Within the sequence of themature protein, beginning at residue E37, the identity between NDS-1 andthe bovine 30-kDa subunit is 95%. As illustrated by FIGS. 9A and 9B,NDS-1 and the bovine 30-kDa subunit have similar hydrophobicity plots.Northern analysis indicates that transcripts of the gene encoding NDS-1are most abundant in cDNA libraries from cancerous tissues andimmortalized cell lines (21/64), in tissues associated with the immunesystem (12/64), in smooth muscle tissues (14/64) and in brain tissue(9/64).

In another embodiment, the invention encompasses a polypeptidecomprising the amino acid sequence of SEQ ID NO:3, as shown in FIGS. 2Aand 2B. NDS-2 is 129 amino acids in length. As shown in FIG. 6, NDS-2has chemical and structural homology with the bovine B15 subunit (GI114). In particular, NDS-2 and 15-kDa (IP) share 74% identity. B15 isone of several nuclear encoded NADH-D subunits that lacks an N-terminalsignal sequence, but is N-acetylated at an adjacent alanine or serineresidue following removal of the initiator methionine. Both NDS-2 and B15 share the critical serine residue in position 2. As illustrated byFIGS. 10A and 10B, NDS-2 and B15 have rather similar hydrophobicityplots. In particular NDS-2 and B15 share a peak of hydrophobicitybetween approximately residues 90 to 115 that is believed to be amembrane spanning alpha-helix. Northern analysis indicates that partialtranscripts of the gene encoding NDS-1 are most abundant in cDNAlibraries from cancerous tissues (26/81), particularly in prostate,brain, and bladder tumors, and in smooth muscle tissues (26/81). It isalso notable in fetal and neonatal tissues, the brain and spinal cord,and in cells of the immune system.

In another embodiment, the invention encompasses a polypeptidecomprising the amino acid sequence of SEQ ID NO:5, as shown in FIGS. 3Aand 3B NDS-3 is 106 amino acids in length. As shown in FIG. 7, NDS-3 haschemical and structural homology with the bovine 15-kDa (IP) subunit (GI224). In particular, NDS-3 and 15-kDa (IP) share 75% identity. The15-kDa subunit is one of several subunits found in the iron-suifirprotein (IP) fraction during purification of NADH-D. The 15-kDa subunitcontains four cysteine residues, all of which are shared by NDS-3, thatmay form one of the iron-sulfur centers that functions in electrontransport. As illustrated by FIGS. 11A and 11B, NDS-3 and 15-kDa (IP)have rather similar hydrophobicity plots. Northern analysis indicatesthat partial transcripts of the gene encoding NDS-3 are most abundant incDNA libraries from cancerous tissues and immortalized cell lines(28/88), smooth muscle tissues (17/88), and in brain and neural tissues(10/88). They are also found in fetal, developing tissues and in tissuesassociated with inflammation and the immune response.

In another embodiment, the invention encompasses a polypeptidecomprising the amino acid sequence of SEQ ID NO:7, as shown in FIGS. 4Aand 4B NDS-4 is 119 amino acids in length. As shown in FIG. 8, NDS-4 haschemical and structural homology with the bovine 14.5b subunit (GI 582).In particular, NDS-4 and 14.5b share 71% identity. The bovine 14.5bsubunit is the only one of the nine nuclear encoded subunits that doesnot contain an alanine or serine residue adjacent to the initiatormethionine and appears to be N-acetylated at the N-terminus withoutremoval of the initiator methionine. NDS-4 shares this feature. Asillustrated by FIGS. 12A and 12B, NDS-4 and 14.5b share similarhydrophobicity plots. Neither protein is notably hydrophobic, which isconsistent with the occurrence of 14.5b in the extrinsic membrane domainof NADH-D. Northern analysis indicates that partial transcripts of thegene encoding NDS-4 are most abundant in cDNA libraries from canceroustissues and immortalized cell lines (28/70) and smooth muscle tissues(19/70), with some occurrence in fetal, developing tissues as well.

The invention also encompasses NDS variants. A preferred NDS variant isone having at least 80%, and more preferably 90%, amino acid sequencesimilarity to the NDS amino acid sequence (SEQ ID NO:1, SEQ ID NO:3, SEQID NO:5, or SEQ ID NO:7). A most preferred NDS variant is one having atleast 95% amino acid sequence similarity to SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:5, or SEQ ID NO:7.

The invention also encompasses polynucleotides which encode NDS.Accordingly, any nucleic acid sequence which encodes the amino acidsequence of NDS can be used to generate recombinant molecules whichexpress NDS. In a particular embodiment, the invention encompasses thepolynucleotide comprising the nucleic acid of SEQ ID NO:2, SEQ ID NO:4,SEQ ID NO:6, or SEQ ID NO:8 as shown in FIGS. 1A and 1B, 2, and 3.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of nucleotide sequencesencoding NDS, some bearing minimal homology to the nucleotide sequencesof any known and naturally occurring gene, may be produced. Thus, theinvention contemplates each and every possible variation of nucleotidesequence that could be made by selecting combinations based on possiblecodon choices. These combinations are made in accordance with thestandard triplet genetic code as applied to the nucleotide sequence ofnaturally occurring NDS, and all such variations are to be considered asbeing specifically disclosed.

Although nucleotide sequences which encode NDS and its variants arepreferably capable of hybridizing to the nucleotide sequence of thenaturally occurring NDS under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding NDS or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding NDS and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

The invention also encompasses production of DNA sequences, or portionsthereof, which encode NDS and its derivatives, entirely by syntheticchemistry. After production, the synthetic sequence may be inserted intoany of the many available expression vectors and cell systems usingreagents that are well known in the art at the time of the filing ofthis application. Moreover, synthetic chemistry may be used to introducemutations into a sequence encoding NDS or any portion thereof

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridizing to the claimed nucleotide sequences, and inparticular, those shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQID NO:8, under various conditions of stringency. Hybridizationconditions are based on the melting temperature (Tm) of the nucleic acidbinding complex or probe, as taught in Wahl, G. M. and S. L. Berger(1987; Methods Enzymol. 152:399-407) and Kimmel, A. R. (1987; MethodsEnzymol. 152:507-11), and may be used at a defined stringency.

Altered nucleic acid sequences encoding NDS which are encompassed by theinvention include deletions, insertions, or substitutions of differentnucleotides resulting in a polynucleotide that encodes the same or afunctionally equivalent NDS. The encoded protein may also containdeletions, insertions, or substitutions of amino acid residues whichproduce a silent change and result in a functionally equivalent NDS.Deliberate amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe biological activity of NDS is retained. For example, negativelycharged amino acids may include aspartic acid and glutamic acid;positively charged amino acids may include lysine and arginine; andamino acids with uncharged polar head groups having similarhydrophilicity values may include leucine, isoleucine, and valine;glycine and alanine; asparagine and glutamine; serine and threonine;phenylalanine and tyrosine.

Also included within the scope of the present invention are alleles ofthe gene encoding NDS. As used herein, an “allele” or “allelic sequence”is an alternative form of the gene which may result from at least onemutation in the nucleic acid sequence. Alleles may result in alteredmRNAs or polypeptides whose structure or function may or may not bealtered. Any given gene may have none, one, or many allelic forms.Common mutational changes which give rise to alleles are generallyascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

Methods for DNA sequencing which are well known and generally availablein the art may be used to practice any embodiments of the invention. Themethods may employ such enzymes as the Klenow fragment of DNA polymeraseI, SEQUENASE® (US Biochemical Corp, Cleveland, Ohio), Taq polymerase(Perkin Elmer), thermostable T7 polymerase (Amersham, Chicago, Ill.), orcombinations of recombinant polymerases and proofreading exonucleasessuch as the ELONGASE amplification system marketed by Gibco BRL(Gaithersburg, Md.). Preferably, the process is automated with machinessuch as the Hamilton MICROLAB 2200 (Hamilton, Reno, Nev.), PeltierThermal Cycler (PTC200; MJ Research, Watertown, Mass.) and the ABI 377DNA sequencers (Perkin Elmer).

The nucleic acid sequences encoding NDS may be extended utilizing apartial nucleotide sequence and employing various methods known in theart to detect upstream sequences such as promoters and regulatoryelements. For example, one method which may be employed,“restriction-site” PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Sarkar, G. (1993) PCR MethodsApplic. 2:318-322). In particular, genomic DNA is first amplified in thepresence of primer to linker sequence and a primer specific to the knownregion. The amplified sequences are then subjected to a second round ofPCR with the same linker primer and another specific primer internal tothe first one. Products of each round of PCR are transcribed with anappropriate RNA polymerase and sequenced using reverse transcriptase.

Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia, T. et al. (1988)Nucleic Acids Res. 16:8186). The primers may be designed using OLIGOprimer analysis software (National Biosciences Inc., Plymouth, Minn.),or another appropriate program, to be 22-30 nucleotides in length, tohave a GC content of 50% or more, and to anneal to the target sequenceat temperatures about 68°-72° C. The method uses several restrictionenzymes to generate a suitable fragment in the known region of a gene.The fragment is then circularized by intramolecular ligation and used asa PCR template.

Another method which may be used is capture PCR which involves PCRamplification of DNA fragments adjacent to a known sequence in human andyeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCRMethods Applic. 1:111-119). In this method, multiple restriction enzymedigestions and ligations may also be used to place an engineereddouble-stranded sequence into an unknown portion of the DNA moleculebefore performing PCR.

Another method which may be used to retrieve unknown sequences is thatof Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060).Additionally, one may use PCR, nested primers, and PROMOTERFINDERlibraries to walk in genomic DNA (Clontech, Palo Alto, Calif.). Thisprocess avoids the need to screen libraries and is useful in findingintron/exon junctions.

When screening for full-length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable in that they will contain moresequences which contain the 5′ regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariesmay be useful for extension of sequence into the 5′ and 3′non-transcribed regulatory regions.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentfluorescent dyes (one for each nucleotide) which are laser activated,and detection of the emitted wavelengths by a charge coupled devicecamera. Output/light intensity may be converted to electrical signalusing appropriate software (e.g. GENOTYPER and SEQUENCE NAVIGATOR,Perkin Elmer) and the entire process from loading of samples to computeranalysis and electronic data display may be computer controlled.Capillary electrophoresis is especially preferable for the sequencing ofsmall pieces of DNA which might be present in limited amounts in aparticular sample.

In another embodiment of the invention, polynucleotide sequences orfragments thereof which encode NDS, or fusion proteins or functionalequivalents thereof, may be used in recombinant DNA molecules to directexpression of NDS in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced and these sequences may be used to clone and expressNDS.

As will be understood by those of skill in the art, it may beadvantageous to produce NDS-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce a recombinant RNAtranscript having desirable properties, such as a half-life which islonger than that of a transcript generated from the naturally occurringsequence.

The nucleotide sequences of the present invention can be engineeredusing methods generally known in the art in order to alter sequencesencoding NDS for a variety of reasons, including but not limited to,alterations which modify the cloning, processing, and/or expression ofthe gene product. DNA shuffling by random fragmentation and PCRreassembly of gene fragments and synthetic oligonucleotides may be usedto engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, to alterglycosylation patterns, to change codon preference, to produce splicevariants, or to introduce mutations, and so forth.

In another embodiment of the invention, natural, modified, orrecombinant polynucleotides encoding NDS may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of NDS activity, it may be useful toencode a chimeric NDS protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between a sequence encoding NDS and theheterologous protein sequence, so that NDS may be cleaved and purifiedaway from the heterologous moiety.

In another embodiment, sequences encoding NDS may be synthesized, inwhole or in part, using chemical methods well known in the art (seeCaruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223,Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232).Alternatively, the protein itself may be produced using chemical methodsto synthesize the amino acid sequence of NDS, or a portion thereof. Forexample, peptide synthesis can be performed using various solid-phasetechniques (Roberge, J. Y. et al. (1995) Science 269:202-204) andautomated synthesis may be achieved, for example, using the ABI 431Apeptide synthesizer (Perkin Elmer).

The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton, T.(1983) Proteins, Structures and Molecular Principles, W H Freeman andCo., New York, N.Y.). The composition of the synthetic peptides may beconfirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure; Creighton, supra). Additionally, the amino acidsequence of NDS, or any part thereof, may be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

In order to express a biologically active NDS, the nucleotide sequencesencoding NDS or functional equivalents, may be inserted into appropriateexpression vectors, i.e., a vector which contains the necessary elementsfor the transcription and translation of the inserted coding sequence.

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing sequences encoding NDS andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed in Sambrook, J. et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. etal. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.

A variety of expression vector/host systems may be utilized to containand express sequences encoding NDS. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

The “control elements” or “regulatory sequences” are thosenon-translated regions of the vector—enhancers, promoters, 5′ and 3′untranslated regions—which interact with host cellular proteins to carryout transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene,LaJolla, Calif.) or PSPORT1 plasmid (Gibco BRL), and the like, may beused. The baculovirus polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO; and storage protein genes) or from plant viruses(e.g., viral promoters or leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequence encoding NDS,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for NDS. For example, when largequantities of NDS are needed for the induction of antibodies, vectorswhich direct high level expression of fusion proteins that are readilypurified may be used. Such vectors include, but are not limited to, themultifunctional E. coli cloning and expression vectors such asBluescript® (Stratagene), in which the sequence encoding NDS may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster(1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors(Promega, Madison, Wis.) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

In the yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used. For reviews, see Ausubel et al. (supra)and Grant et al. (1987) Methods Enzymol. 153:516-544.

In cases where plant expression vectors are used, the expression of asequence encoding NDS may be driven by any of a number of promoters. Forexample, viral promoters such as the 35S and 19S promoters of CaMV maybe used alone or in combination with the omega leader sequence from TMV(Takamatsu, N. (1987) EMBO J. 6:307-311). Alternatively, plant promoterssuch as the small subunit of RUBISCO or heat shock promoters may be used(Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al.(1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl.Cell Differ. 17:85-105). These constructs can be introduced into plantcells by direct DNA transformation or pathogen-mediated transfection.Such techniques are described in a number of generally available reviews(see, for example, Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook ofScience and Technology (1992) McGraw Hill, New York, N.Y.; pp. 191-196).

An insect system may also be used to express NDS. For example, in onesuch system, Autographa californica nuclear polyhedrosis virus (AcNPV)is used as a vector to express foreign genes in Spodoptera frugiperdacells or in Trichoplusia larvae. The sequences encoding NDS may becloned into a non-essential region of the virus, such as the polyhedringene, and placed under control of the polyhedrin promoter. Successfulinsertion of NDS will render the polyhedrin gene inactive and producerecombinant virus lacking coat protein. The recombinant viruses may thenbe used to infect, for example, S. frugiperda cells or Trichoplusialarvae in which NDS may be expressed (Engelhard, E. K. et al. (1994)Proc. Nat. Acad. Sci. 91:3224-3227).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, sequences encoding NDS may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain a viable virus which iscapable of expressing NDS in infected host cells (Logan, J. and Shenk,T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding NDS. Such signals include the ATGinitiation codon and adjacent sequences. In cases where sequencesencoding NDS, its initiation codon, and upstream sequences are insertedinto the appropriate expression vector, no additional transcriptional ortranslational control signals may be needed. However, in cases whereonly coding sequence, or a portion thereof, is inserted, exogenoustranslational control signals including the ATG initiation codon shouldbe provided. Furthermore, the initiation codon should be in the correctreading frame to ensure translation of the entire insert. Exogenoustranslational elements and initiation codons may be of various origins,both natural and synthetic. The efficiency of expression may be enhancedby the inclusion of enhancers which are appropriate for the particularcell system which is used, such as those described in the literature(Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, HEK293, andW138, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, may be chosen toensure the correct modification and processing of the foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressNDS may be transformed using expression vectors which may contain viralorigins of replication and/or endogenous expression elements and aselectable marker gene on the same or on a separate vector. Followingthe introduction of the vector, cells may be allowed to grow for 1-2days in an enriched media before they are switched to selective media.The purpose of the selectable marker is to confer resistance toselection, and its presence allows growth and recovery of cells whichsuccessfully express the introduced sequences. Resistant clones ofstably transformed cells may be proliferated using tissue culturetechniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adeninephosphoribosyltransferase (Lowy, I. et al. (1980) Cell 22:817-23) geneswhich can be employed in tk⁻ or aprt⁻ cells, respectively. Also,antimetabolite, antibiotic or herbicide resistance can be used as thebasis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.77:3567-70); npt, which confers resistance to the aminoglycosidesneomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1-14) and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).Recently, the use of visible markers has gained popularity with suchmarkers as anthocyanins, β glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression mayneed to be confirmed. For example, if the sequence encoding NDS isinserted within a marker gene sequence, recombinant cells containingsequences encoding NDS can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with asequence encoding NDS under the control of a single promoter. Expressionof the marker gene in response to induction or selection usuallyindicates expression of the tandem gene as well.

Alternatively, host cells which contain sequences encoding andexpressing NDS may be identified by a variety of procedures known tothose of skill in the art. These procedures include, but are not limitedto, DNA-DNA or DNA-RNA hybridizations and protein bioassay orimmunoassay techniques which include membrane, solution, or chip basedtechnologies for the detection and/or quantification of the nucleic acidor protein.

The presence of polynucleotide sequences encoding NDS can be detected byDNA-DNA or DNA-RNA hybridization or amplification using probes orportions or fragments of polynucleotides encoding NDS. Nucleic acidamplification based assays involve the use of oligonucleotides oroligomers based on the sequences encoding NDS to detect transformantscontaining DNA or RNA encoding NDS. As used herein “oligonucleotides” or“oligomers” refer to a nucleic acid sequence of at least about 10nucleotides and as many as about 60 nucleotides, preferably about 15 to30 nucleotides, and more preferably about 20-25 nucleotides, which canbe used as a probe or amplimer.

A variety of protocols for detecting and measuring the expression ofNDS, using either polyclonal or monoclonal antibodies specific for theprotein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson NDS is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, Laborato Manual, APS Press, St Paul,Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med. 158:1211-1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding NDS includeoligolabeling, nick translation, end-labeling or PCR amplification usinga labeled nucleotide. Alternatively, sequences encoding NDS, or anyportion thereof, may be cloned into a vector for the production of anMRNA probe. Such vectors are known in the art, are commerciallyavailable, and may be used to synthesize RNA probes in vitro by additionof an appropriate RNA polymerase such as T7, T3, or SP6 and labelednucleotides. These procedures may be conducted using a variety ofcommercially available kits from Pharmacia & Upjohn (Kalamazoo, Mich.);Promega (Madison, Wis.); and U.S. Biochemical Corp. (Cleveland, Ohio).Suitable reporter molecules or labels, which may be used, includeradionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents as well as substrates, cofactors, inhibitors, magnetic particles,and the like.

Host cells transformed with nucleotide sequences encoding NDS may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a recombinantcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodeNDS may be designed to contain signal sequences which direct secretionof NDS through a prokaryotic or eukaryotic cell membrane. Otherrecombinant constructions may be used to join sequences encoding NDS tonucleotide sequence encoding a polypeptide domain which will facilitatepurification of soluble proteins. Such purification facilitating domainsinclude, but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences such as those specific for Factor XA orenterokinase (Invitrogen, San Diego, Calif.) between the purificationdomain and NDS may be used to facilitate purification. One suchexpression vector provides for expression of a fusion protein containingNDS and a nucleic acid encoding 6 histidine residues preceding athioredoxin or an enterokinase cleavage site. The histidine residuesfacilitate purification on IMIAC (immobilized metal ion affinitychromatography) as described in Porath, J. et al. (1992, Prot. Exp.Purif. 3: 263-281) while the enterokinase cleavage site provides a meansfor purifying NDS from the fusion protein. A discussion of vectors whichcontain fusion proteins is provided in Kroll, D. J. et al. (1993; DNACell Biol. 12:441-453).

In addition to recombinant production, fragments of NDS may be producedby direct peptide synthesis using solid-phase techniques (Merrifield J.(1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesis may beperformed using manual techniques or by automation. Automated synthesismay be achieved, for example, using Applied Biosystems 431A peptidesynthesizers (Perkin Elmer). Various fragments of NDS may be chemicallysynthesized separately and combined using chemical methods to producethe full length molecule.

Therapeutics

Based on the chemical and structural homology between NDS-1 and thebovine 30-kDa subunit, and the expression of NDS-1 in cancerous tissuesand tissues of the immune system, NDS-1 is believed to play a role incancer, the development of inflammatory disease and immunologicaldisorders.

Therefore, in one embodiment, a vector expressing antisense of thepolynucleotide encoding NDS-1 may be administered to a subject to treator prevent cancer. Cancers may include, but are not limited to, cancersof the lung, colon, prostate, heart, brain, stomach, intestine,pancreas, breast, ovaries, leukemias and melanomas.

In another embodiment, a vector expressing antisense of thepolynucleotide encoding NDS-1 may be administered to a subject to treator prevent immune system disorders, which may include, but are notlimited to conditions such as anemias, asthma, systemic lupus,myasthenia gravis, diabetes mellitus, autoimmune thyroiditis,pancreatitis, ulcerative colitis, osteoporosis, glomerulonephritis;rheumatoid and osteoarthritis; and scleroderma.

In another embodiment, antagonists or inhibitors of NDS-1 may beadministered to a subject to treat or prevent cancer, the types ofcancer include those described above.

In another embodiment, antagonists or inhibitors of NDS-1 may beadministered to a subject to treat or prevent the immune systemdisorders listed above.

Based on the chemical and structural homology between NDS-2 and thebovine B15 subunit, and the expression of NDS-2 in cancerous tissues andtissues of the sympathetic nervous system (paraganglion and smoothmuscle tissues), NDS-2 is believed to play a role in cancers,mitochondrial myopathies, and disorders of the sympathetic nervoussystem.

Therefore, in one embodiment, NDS-2 or a fragment or derivative thereofmay be administered to a subject to treat or prevent mitochondrialmyopathies. Such conditions and diseases may include, but are notlimited to, progressive external ophthalmoplegia, Kearns-Sayre syndrome,myoclonic epilepsy, encephalopathy, cardiomyopathy, and lactic acidosis.

In another embodiment, a vector capable of expressing NDS-2, or afragment or a derivative thereof, may also be administered to a subjectto treat or prevent the mitochondrial myopathies listed above.

In another embodiment, a vector expressing antisense of thepolynucleotide encoding NDS-2 may be administered to a subject to treator prevent cancer. Cancers may include, but are not limited to, cancersof the heart, ovaries, colon, kidney, bladder, prostate, pancreas,brain, stomach, breast, lung, liver, and leukemias.

In another embodiment, a vector expressing antisense of thepolynucleotide encoding NDS-2 may be administered to a subject to treator prevent disorders of the sympathetic nervous system. Such disordersand diseases may include, but are not limited to, hypertension,cardiovascular shock, arrhythmias, asthma, migraine headaches, andanaphylactic shock.

In another embodiment, antagonists or inhibitors of NDS-2 may beadministered to a subject to treat or prevent the types of cancer listedabove.

In another embodiment, antagonists or inhibitors of NDS-2 may beadministered to a subject to treat or prevent the disorders of thesympathetic nervous system listed above.

Based on the chemical and structural homology between NDS-3 and thebovine 15-kDa (IP) subunit, and the expression of NDS-2 in canceroustissues, smooth muscle tissues and brain, NDS-3 is believed to play arole in cancer, myopathies, and neurodegenerative diseases.

Therefore, in one embodiment, NDS-3 or a fragment or derivative thereofmay be administered to a subject to treat or prevent mitochondrialmyopathies. Such conditions and diseases may include, but are notlimited to, progressive external ophthalmoplegia, Kearns-Sayre syndrome,myoclonic epilepsy, encephalopathy, cardiomyopathy, and lactic acidosis.

In another embodiment, NDS-3 or a fragment or derivative thereof may beadministered to a subject to treat or prevent neurodegenerativediseases. Such diseases and disorders may include, but are not limitedto, Alzheimer's disease, Huntington's disease, Parkinson's disease,epilepsy, and Down's syndrome,

In another embodiment, a vector capable of expressing NDS-3, or afragment or a derivative thereof, may also be administered to a subjectto treat or prevent myopathies as described above.

In another embodiment, a vector capable of expressing NDS-3, or afragment or a derivative thereof, may also be administered to a subjectto treat or prevent neurodegenerative diseases as described above.

In another embodiment, a vector expressing antisense of thepolynucleotide encoding NDS-3 may be administered to a subject to treator prevent cancers. Cancers may include, but are not limited to, cancersof the ovaries, paraganglion, uterus, kidney, brain, heart, prostate,stomach, bladder, lung, colon, heart, tongue, thyroid, and breast.

In another embodiment, antagonists or inhibitors of NDS-3 may beadministered to a subject to treat or prevent cancers as describedabove.

Based on the chemical and structural homology between NDS-4 (SEQ IDNO:7) and the bovine B14.5b subunit (SEQ ID NO:12), and the expressionof NDS-4 in cancerous tissues, and tissues of the sympathetic nervoussystem (paraganglion and smooth muscle tissues), NDS-4 is believed toplay a role in cancer, mitochondrial myopathies, and disorders of thesympathetic nervous system.

Therefore, in one embodiment, NDS-4 or a fragment or derivative thereofmay be administered to a subject to treat or prevent mitochondrialmyopathies. Such conditions and diseases may include, but are notlimited to, progressive external ophthalmoplegia, Kearns-Sayre syndrome,myoclonic epilepsy, encephalopathy, cardiomyopathy, and lactic acidosis.

In another embodiment, a vector capable of expressing NDS-4, or afragment or a derivative thereof, may also be administered to a subjectto treat or prevent myopathies as described above.

In another embodiment, a vector expressing antisense of thepolynucleotide encoding NDS-4 may be administered to a subject to treator prevent cancer. Cancers may include, but are not limited to, cancersof the heart, ovaries, colon, kidney, bladder, prostate, pancreas,brain, stomach, breast, lung, liver, and leukemias.

In another embodiment, a vector expressing antisense of thepolynucleotide encoding NDS-4 may be administered to a subject to treator prevent disorders of the sympathetic nervous system. Such disordersand diseases may include, but are not limited to, hypertension,cardiovascular shock, arrhythmias, asthma, migraine headaches, andanaphylactic shock.

In another embodiment, antagonists or inhibitors of NDS-4 may beadministered to a subject to treat or prevent the types of cancer listedabove.

In another embodiment, antagonists or inhibitors of NDS-4 may beadministered to a subject to treat or prevent the disorders of thesympathetic nervous system listed above.

Antibodies which are specific for NDS may be used directly as anantagonist, or indirectly as a targeting or delivery mechanism forbringing a pharmaceutical agent to cells or tissue which express NDS.

In other embodiments, any of the therapeutic proteins, antagonists,antibodies, agonists, antisense sequences or vectors described above maybe administered in combination with other appropriate therapeuticagents. Selection of the appropriate agents for use in combinationtherapy may be made by one of ordinary skill in the art, according toconventional pharmaceutical principles. The combination of therapeuticagents may act synergistically to effect the treatment or prevention ofthe various disorders described above. Using this approach, one may beable to achieve therapeutic efficacy with lower dosages of each agent,thus reducing the potential for adverse side effects.

Antagonists or inhibitors of NDS may be produced using methods which aregenerally known in the art. In particular, purified NDS may be used toproduce antibodies or to screen libraries of pharmaceutical agents toidentify those which specifically bind NDS.

Antibodies to NDS may be generated using methods that are well known inthe art. Such antibodies may include, but are not limited to,polyclonal, monoclonal, chimeric, single chain, Fab fragments, andfragments produced by a Fab expression library. Neutralizing antibodies,(i.e., those which inhibit dimer formation) are especially preferred fortherapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith NDS or any fragment or oligopeptide thereof which has immunogenicproperties. Depending on the host species, various adjuvants may be usedto increase immunological response. Such adjuvants include, but are notlimited to, Freund's, mineral gels such as aluminum hydroxide, andsurface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially preferable.

It is preferred that the peptides, fragments, or oligopeptides used toinduce antibodies to NDS have an amino acid sequence consisting of atleast five amino acids, and more preferably at least 10 amino acids. Itis also preferable that they are identical to a portion of the aminoacid sequence of the natural protein, and they may contain the entireamino acid sequence of a small, naturally occurring molecule. Shortstretches of NDS amino acids may be fused with those of another proteinsuch as keyhole limpet hemocyanin and antibody produced against thechimeric molecule.

Monoclonal antibodies to NDS may be prepared using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the EBV-hybridomatechnique (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. etal. (1985) J Immunol. Methods 81:31-42; Cote, R. J. et al. (1983) Proc.Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al. (1985) Mol Cell Biol.61:109-120).

In addition, techniques developed for the production of “chimericantibodies”, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison, S. L. et al. (1984) Proc.Natl. Acad. Sci. 81:6851-55; Neuberger, M. S. et al. (1984) Nature312:604-8; Takeda, S. et al. (1985) Nature 314:452-4). Alternatively,techniques described for the production of single chain antibodies maybe adapted, using methods known in the art, to produce NDS-specificsingle chain antibodies. Antibodies with related specificity, but ofdistinct idiotypic composition, may be generated by chain shuffling fromrandom combinatorial immunoglobin libraries (Burton D. R. (1991) Proc.Natl. Acad. Sci. 88:11120-3).

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inthe literature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86:3833-37; Winter, G. et al. (1991) Nature 349:293-9).

Antibody fragments which contain specific binding sites for NDS may alsobe generated. For example, such fragments include, but are not limitedto, the F(ab′)2 fragments which can be produced by pepsin digestion ofthe antibody molecule and the Fab fragments which can be generated byreducing the disulfide bridges of the F(ab′)2 fragments. Alternatively,Fab expression libraries may be constructed to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificity(Huse, W. D. et al. (1989) Science 254:1275-81).

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between NDS and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering NDS epitopes is preferred, but a competitive bindingassay may also be employed (Maddox, supra).

In another embodiment of the invention, the polynucleotides encodingNDS, or any fragment thereof, or antisense molecules, may be used fortherapeutic purposes. In one aspect, antisense to the polynucleotideencoding NDS may be used in situations in which it would be desirable toblock the transcription of mRNA. In particular, cells may be transformedwith sequences complementary to polynucleotides encoding NDS. Thus,antisense sequences may be used to modulate NDS activity, or to achieveregulation of gene function. Such technology is now well known in theart, and sense or antisense oligomers or larger fragments, can bedesigned from various locations along the coding or control regions ofsequences encoding NDS.

Expression vectors derived from retroviruses, adenovirus, herpes orvaccinia viruses, or from various bacterial plasmids may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct recombinant vectors which will express antisensepolynucleotides of the gene encoding NDS. These techniques are describedboth in Sambrook et al. (supra) and in Ausubel et al. (supra).

Genes encoding native NDS can be turned off by transforming a cell ortissue with expression vectors which express high levels of thepolynucleotide, or fragment thereof, which encodes NDS. Such constructsmay be used to introduce untranslatable sense or antisense sequencesinto a cell. Even in the absence of integration into the genomic DNA,such vectors may continue to transcribe RNA molecules until they aredisabled by endogenous nucleases. Transient expression may last for amonth or more with a non-replicating vector and even longer ifappropriate replication elements are part of the vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning antisense molecules, DNA, RNA, or PNA, to the control regionsof the gene encoding NDS, i.e., the promoters, enhancers, and introns.Oligonucleotides derived from the transcription initiation site, e.g.,between positions −10 and +10 from the start site, are preferred.Similarly, inhibition can be achieved using “triple helix” base-pairingmethodology. Triple helix pairing is useful because it causes inhibitionof the ability of the double helix to open sufficiently for the bindingof polymerases, transcription factors, or regulatory molecules. Recenttherapeutic advances using triplex DNA have been described in theliterature (Gee, J. E. et al. (1994) In: Huber, B. E. and B. I. Carr,Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco,N.Y.). The antisense molecules may also be designed to block translationof mRNA by preventing the transcript from binding to ribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Exampleswhich may be used include engineered hammerhead motif ribozyme moleculesthat can specifically and efficiently catalyze endonucleolytic cleavageof sequences encoding NDS.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Antisense molecules and ribozymes of the invention may be prepared byany method known in the art for the synthesis of nucleic acid molecules.These include techniques for chemically synthesizing oligonucleotidessuch as solid phase phosphoramidite chemical synthesis. Alternatively,RNA molecules may be generated by in vitro and in vivo transcription ofDNA sequences encoding NDS. Such DNA sequences may be incorporated intoa wide variety of vectors with suitable RNA polymerase promoters such asT7 or SP6. Alternatively, these cDNA constructs that synthesizeantisense RNA constitutively or inducibly can be introduced into celllines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5′ and/or 3′ ends of the moleculeor the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection and by liposome injections may beachieved using methods which are well known in the art.

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such asdogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administrationof a pharmaceutical composition, in conjunction with a pharmaceuticallyacceptable carrier, for any of the therapeutic effects discussed above.Such pharmaceutical compositions may consist of NDS, antibodies to NDS,mimetics, agonists, antagonists, or inhibitors of NDS. The compositionsmay be administered alone or in combination with at least one otheragent, such as stabilizing compound, which may be administered in anysterile, biocompatible pharmaceutical carrier, including, but notlimited to, saline, buffered saline, dextrose, and water. Thecompositions may be administered to a patient alone, or in combinationwith other agents, drugs or hormones.

The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gurn arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks's solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils such as sesame oil, or synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Optionally, the suspensionmay also contain suitable stabilizers or agents which increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at apH range of 4.5 to 5.5, that is combined with buffer prior to use.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. For administration of NDS, such labeling would includeamount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models, usually mice, rabbits, dogs, or pigs. The animal modelmay also be used to determine the appropriate concentration range androute of administration. Such information can then be used to determineuseful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example NDS or fragments thereof, antibodies of NDS,agonists, antagonists or inhibitors of NDS, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio between therapeutic and toxic effects is thetherapeutic index, and it can be expressed as the ratio, ED50/LD50.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

Diagnostics

In another embodiment, antibodies which specifically bind NDS may beused for the diagnosis of conditions or diseases characterized byexpression of NDS, or in assays to monitor patients being treated withNDS, agonists, antagonists or inhibitors. The antibodies useful fordiagnostic purposes may be prepared in the same manner as thosedescribed above for therapeutics. Diagnostic assays for NDS includemethods which utilize the antibody and a label to detect NDS in humanbody fluids or extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by joining them, eithercovalently or non-covalently, with a reporter molecule. A wide varietyof reporter molecules which are known in the art may be used, several ofwhich are described above.

A variety of protocols including ELISA, RIA, and FACS for measuring NDSare known in the art and provide a basis for diagnosing altered orabnormal levels of NDS expression. Normal or standard values for NDSexpression are established by combining body fluids or cell extractstaken from normal mammalian subjects, preferably human, with antibody toNDS under conditions suitable for complex formation The amount ofstandard complex formation may be quantified by various methods, butpreferably by photometric, means. Quantities of NDS expressed in subjectsamples, control and disease, from biopsied tissues are compared withthe standard values. Deviation between standard and subject valuesestablishes the parameters for diagnosing disease.

In another embodiment of the invention, the polynucleotides encoding NDSmay be used for diagnostic purposes. The polynucleotides which may beused include oligonucleotide sequences, antisense RNA and DNA molecules,and PNAs. The polynucleotides may be used to detect and quantitate geneexpression in biopsied tissues in which expression of NDS may becorrelated with disease. The diagnostic assay may be used to distinguishbetween absence, presence, and excess expression of NDS, and to monitorregulation of NDS levels during therapeutic intervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding NDS or closely related molecules, may be used to identifynucleic acid sequences which encode NDS. The specificity of the probe,whether it is made from a highly specific region, e.g., 10 uniquenucleotides in the 5′ regulatory region, or a less specific region,e.g., especially in the 3′ coding region, and the stringency of thehybridization or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding NDS, alleles, or related sequences.

Probes may also be used for the detection of related sequences, andshould preferably contain at least 50% of the nucleotides from any ofthe sequences encoding NDS. The hybridization probes of the subjectinvention may be DNA or RNA and derived from the nucleotide sequences ofSEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:8 or from genomicsequence including promoter, enhancer elements, and introns of thenaturally occurring NDS.

Means for producing specific hybridization probes for DNAs encoding NDSinclude the cloning of nucleic acid sequences encoding NDS or NDSderivatives into vectors for the production of mRNA probes. Such vectorsare known in the art, commercially available, and may be used tosynthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, radionuclides such as 32P or 35S, or enzymatic labels, such asalkaline phosphatase coupled to the probe via avidin/biotin couplingsystems, and the like.

Polynucleotide sequences encoding NDS may be used for the diagnosis ofconditions or diseases which are associated with expression of NDS.Examples of such conditions or diseases include cancers of the heart,breast, colon, and prostate, neurodegenerative diseases such asAlzheimer's and Huntington's disease, immunological disorders such asanemias, asthma, systemic lupus, myasthenia gravis, diabetes mellitus,autoimmune thyroiditis, pancreatitis, ulcerative colitis, osteoporosis,glomerulonephritis; rheumatoid and osteoarthritis; and scleroderma,myopathies such as progressive external ophthalmoplegia, Kearns-Sayresyndrome, myoclonic epilepsy, encephalopathy, cardiomyopathy, and lacticacidosis, and disorders of the sympathetic nervous system such ashypertension, cardiovascular shock, arrhythmias, asthma, migraineheadaches, and anaphylactic shock. The polynucleotide sequences encodingNDS may be used in Southern or northern analysis, dot blot, or othermembrane-based technologies; in PCR technologies; or in dip stick, pin,ELISA or chip assays utilizing fluids or tissues from patient biopsiesto detect altered NDS expression. Such qualitative or quantitativemethods are well known in the art.

In a particular aspect, the nucleotide sequences encoding NDS may beuseful in assays that detect activation or induction of various cancers,particularly those mentioned above. The nucleotide sequences encodingNDS may be labeled by standard methods, and added to a fluid or tissuesample from a patient under conditions suitable for the formation ofhybridization complexes. After a suitable incubation period, the sampleis washed and the signal is quantitated and compared with a standardvalue. If the amount of signal in the biopsied or extracted sample issignificantly altered from that of a comparable control sample, thenucleotide sequences have hybridized with nucleotide sequences in thesample, and the presence of altered levels of nucleotide sequencesencoding NDS in the sample indicates the presence of the associateddisease. Such assays may also be used to evaluate the efficacy of aparticular therapeutic treatment regimen in animal studies, in clinicaltrials, or in monitoring the treatment of an individual patient.

In order to provide a basis for the diagnosis of disease associated withexpression of NDS, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, which encodes NDS, under conditionssuitable for hybridization or amplification. Standard hybridization maybe quantified by comparing the values obtained from normal subjects withthose from an experiment where a known amount of a substantiallypurified polynucleotide is used. Standard values obtained from normalsamples may be compared with values obtained from samples from patientswho are symptomatic for disease. Deviation between standard and subjectvalues is used to establish the presence of disease.

Once disease is established and a treatment protocol is initiated,hybridization assays may be repeated on a regular basis to evaluatewhether the level of expression in the patient begins to approximatethat which is observed in the normal patient. The results obtained fromsuccessive assays may be used to show the efficacy of treatment over aperiod ranging from several days to months.

With respect to cancer, the presence of a relatively low amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding NDS may involve the use of PCR. Such oligomers may bechemically synthesized, generated enzymatically, or produced from arecombinant source. Oligomers will preferably consist of two nucleotidesequences, one with sense orientation (5′→3′) and another with antisense(3′←5′), employed under optimized conditions for identification of aspecific gene or condition. The same two oligomers, nested sets ofoligomers, or even a degenerate pool of oligomers may be employed underless stringent conditions for detection and/or quantitation of closelyrelated DNA or RNA sequences.

Methods which may also be used to quantitate the expression of NDSinclude radiolabeling or biotinylating nucleotides, coamplification of acontrol nucleic acid, and standard curves onto which the experimentalresults are interpolated (Melby, P. C. et al. (1993) J. Immunol.Methods, 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem. 229-236).The speed of quantitation of multiple samples may be accelerated byrunning the assay in an ELISA format where the oligomer of interest ispresented in various dilutions and a spectrophotometric or colorimetricresponse gives rapid quantitation.

In another embodiment of the invention, the nucleic acid sequence whichencodes NDS may also be used to generate hybridization probes which areuseful for mapping the naturally occurring genomic sequence. Thesequence may be mapped to a particular chromosome or to a specificregion of the chromosome using well known techniques. Such techniquesinclude FISH, FACS, or artificial chromosome constructions, such asyeast artificial chromosomes, bacterial artificial chromosomes,bacterial P1 constructions or single chromosome cDNA libraries asreviewed in Price, C. M. (1993) Blood Rev. 7:127-134, and Trask, B. J.(1991) Trends Genet. 7:149-154.

FISH (as described in Verma, R. S. et al. (1988) Human Chromosomes: AManual Qf Basic Techniques, Pergamon Press, New York, N.Y.) may becorrelated with other physical chromosome mapping techniques and geneticmap data. Examples of genetic map data can be found in the 1994 GenomeIssue of Science (265:1981f). Correlation between the location of thegene encoding NDS on a physical chromosomal map and a specific disease,or predisposition to a specific disease, may help delimit the region ofDNA associated with that genetic disease. The nucleotide sequences ofthe subject invention may be used to detect s differences in genesequences between normal, carrier, or affected individuals.

In situ hybridization of chromosomal preparations and physical mappingtechniques such as linkage analysis using established chromosomalmarkers may be used for extending genetic maps. Often the placement of agene on the chromosome of another mammalian species, such as mouse, mayreveal associated markers even if the number or arm of a particularhuman chromosome is not known. New sequences can be assigned tochromosomal arms, or parts thereof, by physical mapping. This providesvaluable information to investigators searching for disease genes usingpositional cloning or other gene discovery techniques. Once the diseaseor syndrome has been crudely localized by genetic linkage to aparticular genomic region, for example, AT to 11q22-23 (Gatti, R. A. etal. (1988) Nature 336:577-580), any sequences mapping to that area mayrepresent associated or regulatory genes for further investigation. Thenucleotide sequence of the subject invention may also be used to detectdifferences in the chromosomal location due to translocation, inversion,etc. among normal, carrier, or affected individuals.

In another embodiment of the invention, NDS, its catalytic orimmunogenic fragments or oligopeptides thereof, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, between NDSand the agent being tested, may be measured.

Another technique for drug screening which may be used provides for highthroughput screening of compounds having suitable binding affinity tothe protein of interest as described in published PCT applicationWO84/03564. In this method, as applied to NDS, large numbers ofdifferent small test compounds are synthesized on a solid substrate,such as plastic pins or some other surface. The test compounds arereacted with NDS, or fragments thereof, and washed. Bound NDS is thendetected by methods well known in the art. Purified NDS can also becoated directly onto plates for use in the aforementioned drug screeningtechniques. Alternatively, non-neutralizing antibodies can be used tocapture the peptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding NDS specificallycompete with a test compound for binding NDS. In this manner, theantibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with NDS.

In additional embodiments, the nucleotide sequences which encode NDS maybe used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

The examples below are provided to illustrate the subject invention andare not included for the purpose of limiting the invention.

EXAMPLES

I cDNA Library Construction

HMC1NOT01

The human mast cell HMC1NOT01 cDNA library was constructed by Stratagene(Stratagene, La Jolla Calif.) using mRNA purified from cultured HMC-1cells. The library was prepared by Stratagene essentially as described.The human mast cell HMC1NOT01 cDNA library was prepared by purifyingpoly(A+)RNA (mRNA) from human mast cells and then enzymaticallysynthesizing double stranded complementary DNA (cDNA) copies of themRNA. Synthetic adaptor oligonucleotides were ligated onto the ends ofthe cDNA enabling its insertion into the lambda vector. The HMC1NOT01library was constructed using the UINZAP vector system (Stratagene),allowing high efficiency unidirectional (sense orientation) lambdalibrary construction and the convenience of a plasmid system withblue/white color selection to detect clones with cDNA insertions.

BLADNOT03

The BLADNOT03 cDNA library was constructed from microscopically normalbladder tissue obtained from a 80-year-old Caucasian female. The normaltissue from the anterior wall was excised along with the tumorous tissueduring a radical cysterectomy of a grade 3 of 4 invasive transitionalcell carcinoma located on the posterior wall. Prior to surgery thepatient had a history of a malignant neoplasm of the uterus, a totalhysterectomy, removal of the fallopian tubes and ovaries, partialthyroidectomy, aorto-coronary bypass, hypertension, and atherosclerosis.The patient was receiving the following medications: COUMADIN (DuPontPharmaceuticals, Wilmington, Pa.), KLOTRIX (Bristol Laboratories,Evansville, Ind.), LASIX (Hoechst-Roussel Pharmaceuticals, Inc.,Somerville, N.J.), digoxin, and atenolol. There was a family history ofatherosclerosis in the father and a sibling, and osteoarthritis in themother.

The frozen tissue was homogenized and lysed using a Brinkmann PolyronPT-3000 homogenizer (Brinkmann Instruments, Westbury, N.J.) inguanidinium isothiocyanate solution. The lysate was centrifuged over a5.7 M CsCl cushion using a Beckman SW28 rotor in a Beckman L8-70MULTRACENTRIFUGE (Beckman Instruments) for 18 hours at 25,000 rpm atambient temperature. The RNA was extracted with acid phenol pH 4.7,precipitated using 0.3 M sodium acetate and 2.5 volumes of ethanol,resuspended in RNAse-free water, and DNase treated at 37° C. The RNAextraction was repeated with acid phenol pH 4.7 and precipitated withsodium acetate and ethanol as before. The mRNA was then isolated usingthe Qiagen OLIGOTEX kit (QIAGEN, Inc.; Chatsworth, Calif.) and used toconstruct the cDNA library.

The mRNA was handled according to the recommended protocols in theSUPERSCRIPT plasmid system for cDNA synthesis and plasmid cloning (Cat.#18248-013, Gibco BRL). A new plasmid was constructed using thefollowing procedures: The commercial plasmid pSPORT 1 (Gibco BRL) wasdigested with Eco RI restriction enzyme (New England Biolabs, Beverley,Mass.), the overhanging ends of the plasmid were filled with Klenowenzyme (New England Biolabs) and 2′-deoxynucleotide-5′-triphosphates(dNTPs), and the intermediate plasmid was self-ligated and transformedinto the bacterial host, E. coli strain JM109.

Quantities of this intermediate plasmid were digested with Hind IIIrestriction enzyme (New England Biolabs), the overhanging ends werefilled with Klenow and dNTPs, and a 10-mer linker of sequence 5′ . . .CGGAATTCCG . . . 3′ was phosphorylated and ligated onto the blunt ends.The product of the ligation reaction was digested with EcoRI andself-ligated. Following transformation into JM109 host cells, plasmidsdesignated pINCY were isolated and tested for the ability to incorporatecDNAs using Not I and Eco RI restriction enzymes.

BLADNOT03 cDNAs were fractionated on a SEPHAROSE CL4B column (Cat.#275105-01, Pharmacia), and those cDNAs exceeding 400 bp were ligatedinto pINCY I. The plasmid pINCY I was subsequently transformed into DH5αcompetent cells (Cat. #18258-012, Gibco BRL).

PROSNOT16

The PROSTNOT16 cDNA library was constructed from microscopically normalprostate obtained from a 68-year-old Caucasian male. The normal prostatetissue was excised during a radical prostatectomy along with prostatetissue for which the pathology report indicated was associated with aGleason grade 3+4 adenocarcinoma which perforated the capsule to involveperiprostatic tissue. Surgical margins (distal urethra, right and leftbladder bases, right and left apices) were negative for tumor.Initially, the patient presented with elevated prostate specific antigen(PSA) after which he was diagnosed with a malignant neoplasm of theprostate and myasthenia gravis. The patient history included benignhypertension, cerebrovascular disease, arteriosclerotic coronary arterydisease, osteoarthritis, type II diabetes without complications, acutemyocardial infarction, and alcohol use. The patient's family historyincluded benign hypertension, an episode of acute myocardial infarction,and hyperlipidemia in the patient's mother, and arterioscleroticcoronary artery disease and an episode of acute myocardial infarction inthe patient's sibling. The patient was taking PREDNISONE (The UpjohnCompany, Kalamazoo, Mich.) and DIABETA (glyburide; Hoechst-RousselPharmaceuticals Inc., Somerville, N.J.).

The frozen tissue was homogenized and lysed using a Brinkmann PolytronPT-3000 Homogenizer (Brinkmann Instruments, Westbury, N.J.) inguanidinium isothiocyanate solution. The lysates were centrifuged over a5.7 M CsCl cushion using a Beckman SW28 rotor in a Beckman L8-70MULTRACENTRIFUGE (Beckman Instruments) for 18 hours at 25,000 rpm atambient temperature. The RNA was extracted with acid phenol pH 4.7,precipitated using 0.3 M sodium acetate and 2.5 volumes of ethanol,resuspended in RNAse-free water, and DNase treated at 37° C. The RNAextraction was repeated with acid phenol pH 4.7 and precipitated withsodium acetate and ethanol as before. The mRNA was then isolated usingthe Qiagen OLIGOTEX kit (QIAGEN, Inc., Chatsworth, Calif.) and used toconstruct the cDNA libraries.

The mRNA was handled according to the recommended protocols in theSUPERSCRIPT plasmid system for cDNA synthesis and plasmid cloning (Cat.#18248-013, Gibco BRL). A new plasmid was constructed using thefollowing procedures: The commercial plasmid pSPORT 1 (Gibco BRL) wasdigested with Eco RI restriction enzyme (New England Biolabs, Beverley,Mass.), the overhanging ends of the plasmid were filled with Klenowenzyme (New England Biolabs) and 2′-deoxynucleotide-5′-triphosphates(dNTPs) ,and the intermediate plasmid was self-ligated and transformedinto the bacterial host, E. coli strain JM109.

Quantities of this intermediate plasmid were digested with Hind IIIrestriction enzyme (New England Biolabs), the overhanging ends werefilled with Klenow and dNTPs, and a 10-mer linker of sequence 5′ . . .CGGAATTCCG . . . 3′ was phosphorylated and ligated onto the blunt ends.The product of the ligation reaction was digested with EcoRI andself-ligated. Following transformation into JM109 host cells, plasmidsdesignated pINCY were isolated and tested for the ability to incorporatecDNAs using Not I and Eco RI restriction enzymes.

BLADNOT03 cDNAs were fractionated on a SEPHAROSE CL4B column (Cat.#275105-01, Pharmacia), and those cDNAs exceeding 400 bp were ligatedinto pINCY I. The plasmid pINCY I was subsequently transformed into DH5αcompetent cells (Cat. #18258-012, Gibco BRL).

BRSTTUT03

The BRSTTUT03 cDNA library was constructed from cancerous breast tissueremoved from a 58-year-old Caucasian female who had undergone unilateralextended simple mastectomy following diagnosis of multicentric invasivegrade 4 mammary lobular carcinoma. The pathology report indicated thattumor cells were identified in the upper outer quadrant of the leftbreast, forming a single predominant mass. Tumor cells were alsoidentified in the lower outer quadrant of the left breast, forming threeseparate nodules. The surgical margins were found negative for tumor.The skin, nipple, and fascia were uninvolved. No evidence of vascularinvasion was found. Eight mid low and two high left axillary lymph nodeswere found negative for tumor. Prior to surgery, the patient wasprescribed tamoxifen to inhibit the induction of mammary carcinoma. Thepatient was also taking ZANTAC (ranitidine hydrochloride; GlaxoWellcome, Inc., Research Triangle Park, N.C.), aspirin, extra-strengthTYLENOL (acetaminophen; McNeil Consumer Products Company, FortWashington, Pa. ), and vitamin C. Prior to surgery, the patient wasdiagnosed with skin cancer, cerebrovascular disease, atherosclerosis,rheumatic heart disease, and osteoarthritis. The patient family historyincluded breast cancer in patient's mother and prostate cancer inpatient's brother. This library is an extension of the cDNA libraryBRSTTUT03.

The frozen tissue was homogenized and lysed using a Brinkmann PolytronPT-3000 homogenizer (Brinkmann Instruments, Westbury, N.J.) inguanidinium isothiocyanate solution. The lysate was centrifuged over a5.7 M CsCl cushion using a Beckman SW28 rotor in a Beckman L8-70MUltracentrifuge (Beckman Instruments) for 18 hours at 25,000 rpm atambient temperature. The RNA was extracted with acid phenol pH 4.7,precipitated using 0.3 M sodium acetate and 2.5 volumes of ethanol,resuspended in RNAse-free water, and DNase treated at 37° C. The RNAextraction was repeated with acid phenol pH 4.7 and precipitated withsodium acetate and ethanol as before. The mRNA was then isolated usingthe Qiagen OLIGOTEX kit (QIAGEN, Inc.; Chatsworth, Calif.) and used toconstruct the cDNA library.

The mRNA was handled according to the recommended protocols in theSUPERSCRIPT plasmid system for cDNA synthesis and plasmid cloning (Cat.#18248-013; Gibco/BRL). cDNAs were fractionated on a SEPHAROSE CL4Bcolumn (Cat. #275105-01; Pharmacia), and those cDNAs exceeding 400 bpwere ligated into pSport I. The plasmid pSport I was subsequentlytransformed into DH12S competent cells (Cat. #18312-017; Gibco/BRL).

II Isolation and Sequencing of cDNA Clones

Plasmid DNA was released from the cells and purified using the R.E.A.L.PREP96 plasmid kit (Catalog #26173; QIAGEN, Inc.). This kit enabled thesimultaneous purification of 96 samples in a 96-well block usingmulti-channel reagent dispensers. The recommended protocol was employedexcept for the following changes: 1) the bacteria were cultured in 1 mlof sterile Terrific Broth (Catalog #22711, LIFE TECHNOLOGIEST™) withcarbenicillin at 25 mg/L and glycerol at 0.4%; 2) after inoculation, thecultures were incubated for 19 hours and at the end of incubation, thecells were lysed with 0.3 ml of lysis buffer; and 3) followingisopropanol precipitation, the plasmid DNA pellet was resuspended in 0.1ml of distilled water. After the last step in the protocol, samples weretransferred to a 96-well block for storage at 4° C.

Alternative methods of purifying plasmid DNA include the use of MAGICMINIPREPS DNA purification system (Cat. No. A7100, Promega) or QIAWELL-8Plasmid, QIAWELL PLUS and QIAWELL ULTRA DNA purification systems(Qiagen, Inc.).

The cDNAs were sequenced by the method of Sanger F. and A. R. Coulson(1975; J. Mol. Biol. 94:441f), using a Hamilton MICROLAB 2200 (Hamilton,Reno Nev.) in combination with four Peltier Thermal Cyclers (PTC200 fromMJ Research, Watertown Mass.) and Applied Biosystems 377 or 373 DNAsequencing systems (Perkin Elmer) and reading frame was determined.

III Homology Searching of cDNA Clones and their Deduced Proteins

Each cDNA was compared to sequences in GenBank using a search algorithmdeveloped by Applied Biosystems and incorporated into the INHERIT-670sequence analysis system. In this algorithm, Pattern SpecificationLanguage (TRW Inc, Los Angeles, Calif.) was used to determine regions ofhomology. The three parameters that determine how the sequencecomparisons run were window size, window offset, and error tolerance.Using a combination of these three parameters, the DNA database wassearched for sequences containing regions of homology to the querysequence, and the appropriate sequences were scored with an initialvalue. Subsequently, these homologous regions were examined using dotmatrix homology plots to distinguish regions of homology from chancematches. Smith-Waterman alignments were used to display the results ofthe homology search.

Peptide and protein sequence homologies were ascertained using theINHERIT- 670 sequence analysis system using the methods similar to thoseused in DNA sequence homologies. Pattern Specification Language andparameter windows were used to search protein databases for sequencescontaining regions of homology which were scored with an initial value.Dot-matrix homology plots were examined to distinguish regions ofsignificant homology from chance matches.

BLAST, which stands for Basic Local Alignment Search Tool (Altschul, S.F. (1993) J. Mol. Evol. 36:290-300; Altschul, S. F. et al. (1990) J.Mol. Biol. 215:403-410), was used to search for local sequencealignments. BLAST produces alignments of both nucleotide and amino acidsequences to determine sequence similarity. Because of the local natureof the alignments, BLAST is especially useful in determining exactmatches or in identifying homologs. BLAST is useful for matches which donot contain gaps. The fundamental unit of BLAST algorithm output is theHigh-scoring Segment Pair (HSP).

An HSP consists of two sequence fragments of arbitrary but equal lengthswhose alignment is locally maximal and for which the alignment scoremeets or exceeds a threshold or cutoff score set by the user. The BLASTapproach is to look for HSPs between a query sequence and a databasesequence, to evaluate the statistical significance of any matches found,and to report only those matches which satisfy the user-selectedthreshold of significance. The parameter E establishes the statisticallysignificant threshold for reporting database sequence matches. E isinterpreted as the upper bound of the expected frequency of chanceoccurrence of an HSP (or set of HSPs) within the context of the entiredatabase search. Any database sequence whose match satisfies E isreported in the program output.

IV Northern Analysis

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound (Sambrook et al., supra).

Analogous computer techniques using BLAST (Altschul, S. F. 1993 and1990, supra) are used to search for identical or related molecules innucleotide databases such as GenBank or the LIFESEQ database (IncytePharmaceuticals, Inc.). This analysis is much faster than multiple,membrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or homologous.

The basis of the search is the product score which is defined as:

% sequence identity×% maximum BLAST score 100

The product score takes into account both the degree of similaritybetween two sequences and the length of the sequence match. For example,with a product score of 40, the match will be exact within a 1-2% error;and at 70, the match will be exact. Homologous molecules are usuallyidentified by selecting those which show product scores between 15 and40, although lower scores may identify related molecules.

The results of northern analysis are reported as a list of libraries inwhich the transcript encoding NDS occurs. Abundance and percentabundance are also reported. Abundance directly reflects the number oftimes a particular transcript is represented in a cDNA library, andpercent abundance is abundance divided by the total number of sequencesexamined in the cDNA library.

V Extension of Polynucleotides Encoding NDS to Full Length or to RecoverRegulatory Sequences

Polynucleotides encoding NDS (SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, orSEQ ID NO:8) are used to design oligonucleotide primers for extending apartial nucleotide sequence to full length or for obtaining 5′ or 3′,intron or other control sequences from genomic libraries. One primer issynthesized to initiate extension in the antisense direction (XLR) andthe other is synthesized to extend sequence in the sense direction(XLF). Primers are used to facilitate the extension of the knownsequence “outward” generating amplicons containing new, unknownnucleotide sequence for the region of interest. The initial primers aredesigned from the cDNA using OLIGO 4.06 software (National Biosciences),or another appropriate program, to be 22-30 nucleotides in length, tohave a GC content of 50% or more, and to anneal to the target sequenceat temperatures about 68°-72° C. Any stretch of nucleotides which wouldresult in hairpin structures and primer-primer dimerizations is avoided.

The original, selected cDNA libraries, or a human genomic library areused to extend the sequence; the latter is most useful to obtain 5′upstream regions. If more extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

By following the instructions for the XL-PCR kit (Perkin Elmer) andthoroughly mixing the enzyme and reaction mix, high fidelityamplification is obtained. Beginning with 40 pmol of each primer and therecommended concentrations of all other components of the kit, PCR isperformed using the Peltier Thermal Cycler (PTC200; M. J. Research,Watertown, Mass.) and the following parameters:

Step 1 94° C. for 1 min (initial denaturation) Step 2 65° C. for 1 minStep 3 68° C. for 6 min Step 4 94° C. for 15 sec Step 5 65° C. for 1 minStep 6 68° C. for 7 min Step 7 Repeat step 4-6 for 15 additional cyclesStep 8 94° C. for 15 sec Step 9 65° C. for 1 min Step 10 68° C. for 7:15min Step 11 Repeat step 8-10 for 12 cycles Step 12 72° C. for 8 min Step13  4° C. (and holding)

A 5-10 μl aliquot of the reaction mixture is analyzed by electrophoresison a low concentration (about 0.6-0.8%) agarose mini-gel to determinewhich reactions were successful in extending the sequence. Bands thoughtto contain the largest products are selected and removed from the gel.Further purification involves using a commercial gel extraction methodsuch as QIAQUICK (QIAGEN Inc., Chatsworth, Calif.). After recovery ofthe DNA, Klenow enzyme is used to trim single-stranded, nucleotideoverhangs creating blunt ends which facilitate religation and cloning.

After ethanol precipitation, the products are redissolved in 13 μl ofligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase are added, and the mixture is incubated at roomtemperature for 2-3 hours or overnight at 16° C. Competent E. coli cells(in 40 μl of appropriate media) are transformed with 3 μl of ligationmixture and cultured in 80 μl of SOC medium (Sambrook et al., supra).After incubation for one hour at 37° C., the whole transformationmixture is plated on Luria Bertani (LB)-agar (Sambrook et al., supra)containing 2×Carb. The following day, several colonies are randomlypicked from each plate and cultured in 150 μl of liquid LB/2×Carb mediumplaced in an individual well of an appropriate, commercially-available,sterile 96-well microtiter plate. The following day, 5 μl of eachovernight culture is transferred into a non-sterile 96-well plate andafter dilution 1:10 with water, 5 μl of each sample is transferred intoa PCR array.

For PCR amplification, 18 μl of concentrated PCR reaction mix (3.3×)containing 4 units of rTth DNA polymerase, a vector primer, and one orboth of the gene specific primers used for the extension reaction areadded to each well. Amplification is performed using the followingconditions:

Step 1 94° C. for 60 sec Step 2 94° C. for 20 sec Step 3 55° C. for 30sec Step 4 72° C. for 90 sec Step 5 Repeat steps 2-4 for an additional29 cycles Step 6 72° C. for 180 sec Step 7  4° C. (and holding)

Aliquots of the PCR reactions are run on agarose gels together withmolecular weight markers. The sizes of the PCR products are compared tothe original partial cDNAs, and appropriate clones are selected, ligatedinto plasmid, and sequenced.

VI Labeling and Use of Hybridization Probes

Hybridization probes derived from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6,or SEQ ID NO:8 are employed to screen cDNAs, genomic DNAs, or mRNAs.Although the labeling of oligonucleotides, consisting of about 20base-pairs, is specifically described, essentially the same procedure isused with larger cDNA fragments. Oligonucleotides are designed usingstate-of-the-art software such as OLIGO 4.06 (National Biosciences),labeled by combining 50 pmol of each oligomer and 250 mCi of [-³²P]adenosine triphosphate (Amersham) and T4 polynucleotide kinase (DuPontNEN®, Boston, Mass.). The labeled oligonucleotides are substantiallypurified with Sephadex G-25 superfine resin column (Pharmacia & Upjohn).A portion containing 10⁷ counts per minute of each of the sense andantisense oligonucleotides is used in a typical membrane basedhybridization analysis of human genomic DNA digested with one of thefollowing endonucleases (Ase I, Bgl II, Eco RI, Pst I, Xba 1, or Pvu II;DuPont NEN®).

The DNA from each digest is fractionated on a 0.7 percent agarose geland transferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham, N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1×salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR™ film(Kodak, Rochester, N.Y.) is exposed to the blots or the blots areexposed in a Phosphoimager cassette (Molecular Dynamics, Sunnyvale,Calif.) hybridization patterns are compared visually.

VII Antisense Molecules

Antisense molecules to the NDS-encoding sequence, or any part thereof,is used to inhibit in vivo or in vitro expression of naturally occurringNDS. Although use of antisense oligonucleotides, comprising about 20base-pairs, is specifically described, essentially the same procedure isused with larger cDNA fragments. An oligonucleotide based on the codingsequences of NDS, as shown in FIGS. 1A, 1B, 1C, 2A, 2B, 3A, 3B, 4A, and4B are used to inhibit expression of naturally occurring NDS. Thecomplementary oligonucleotide is designed from the most unique 5′sequence as shown in FIGS. 1A, 1B, and 1C and used either to inhibittranscription by preventing promoter binding to the upstreamnontranslated sequence or translation of an NDS-encoding transcript bypreventing the ribosome from binding. Using an appropriate portion ofthe signal and 5′ sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, orSEQ ID NO:8, an effective antisense oligonucleotide includes any 15-20nucleotides spanning the region which translates into the signal or 5′coding sequence of the polypeptide as shown in FIGS. 1A, 1B, 1C, 2A, 2B,3A, 3B, 4A, and 4B.

VIII Expression of NDS

Expression of NDS is accomplished by subcloning the cDNAs intoappropriate vectors and transforming the vectors into host cells. Inthis case, the cloning vector, pSport, previously used for thegeneration of the cDNA library is used to express NDS in E. coli.Upstream of the cloning site, this vector contains a promoter forB-galactosidase, followed by sequence containing the amino-terminal Met,and the subsequent seven residues of β-galactosidase. Immediatelyfollowing these eight residues is a bacteriophage promoter useful fortranscription and a linker containing a number of unique restrictionsites.

Induction of an isolated, transformed bacterial strain with IPTG usingstandard methods produces a fusion protein which consists of the firsteight residues of β-galactosidase, about 5 to 15 residues of linker, andthe full length protein. The signal residues direct the secretion of NDSinto the bacterial growth media which can be used directly in thefollowing assay for activity.

IX Demonstration of NDS Activity

NDS activity is measured in the reconsituted NADH-D complex by thecatalysis of electron transfer from NADH to decylubiquinone (DB). Thereaction contains 10 ug/mL NADH-D protein, 2 uM NADH in 50 mM tris-HCLbuffer, pH 7.5, 50 mM NaCl, and 1 mM KCN. The reaction is started byaddition of DB at 2 uM and followed by the change in absorbance at 340nm due to the oxidation of NADH using an ultraviolet spectrophotometer.The activity of NDS is proportional to the rate of change of absorbanceat 340 nm.

X Production of NDS Specific Antibodies

NDS that is substantially purified using PAGE electrophoresis (Sambrook,supra), or other purification techniques, is used to immunize rabbitsand to produce antibodies using standard protocols. The amino acidsequence deduced from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ IDNO:8 is analyzed using DNASTAR software (DNASTAR Inc) to determineregions of high immunogenicity and a corresponding oligopolypeptide issynthesized and used to raise antibodies by means known to those ofskill in the art. Selection of appropriate epitopes, such as those nearthe C-terminus or in hydrophilic regions, is described by Ausubel et al.(supra), and others.

Typically, the oligopeptides are 15 residues in length, synthesizedusing an Applied Biosystems 413A peptide synthesizer usingfmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH, Sigma,St. Louis, Mo.) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimideester (MBS; Ausubel et al., supra). Rabbits are immunized with theoligopeptide-KLH complex in complete Freund's adjuvant. The resultingantisera are tested for antipeptide activity, for example, by bindingthe peptide to plastic, blocking with 1% BSA, reacting with rabbitantisera, washing, and reacting with radioiodinated, goat anti-rabbitIgG.

XI Purification of Naturally Occurring NDS Using Specific Antibodies

Naturally occurring or recombinant NDS is substantially purified byimmunoaffinity chromatography using antibodies specific for NDS. Animmunoaffinity column is constructed by covalently coupling NDS antibodyto an activated chromatographic resin, such as CnBr-activated SEPHAROSE(Pharmacia & Upjohn). After the coupling, the resin is blocked andwashed according to the manufacturer's instructions.

Media containing NDS is passed over the immunoaffinity column, and thecolumn is washed under conditions that allow the preferential absorbanceof NDS (e.g., high ionic strength buffers in the presence of detergent).The column is eluted under conditions that disrupt antibody/NDS binding(eg, a buffer of pH 2-3 or a high concentration of a chaotrope, such asurea or thiocyanate ion), and NDS is collected.

XII Identification of Molecules which Interact with NDS

NDS or biologically active fragments thereof are labeled with ¹²⁵IBolton-Hunter reagent (Bolton, A. E. and W. M. Hunter (1973) Biochem. J.133: 529-39). Candidate molecules previously arrayed in the wells of amulti-well plate are incubated with the labeled NDS, washed and anywells with labeled NDS complex are assayed. Data obtained usingdifferent concentrations of NDS are used to calculate values for thenumber, affinity, and association of NDS with the candidate molecules.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

12 264 amino acids amino acid single linear Consensus Consensus 1 MetAla Ala Ala Ala Val Ala Arg Leu Trp Trp Arg Gly Ile Leu Gly 1 5 10 15Ala Ser Ala Leu Thr Arg Gly Thr Gly Arg Pro Ser Val Leu Leu Leu 20 25 30Pro Val Arg Arg Glu Ser Ala Gly Ala Asp Thr Arg Pro Thr Val Arg 35 40 45Pro Arg Asn Asp Val Ala His Lys Gln Leu Ser Ala Phe Gly Glu Tyr 50 55 60Val Ala Glu Ile Leu Pro Lys Tyr Val Gln Gln Val Gln Val Ser Cys 65 70 7580 Phe Asn Glu Leu Glu Val Cys Ile His Pro Asp Gly Val Ile Pro Val 85 9095 Leu Thr Phe Leu Arg Asp His Thr Asn Ala Gln Phe Lys Ser Leu Val 100105 110 Asp Leu Thr Ala Val Asp Val Pro Thr Arg Gln Asn Arg Phe Glu Ile115 120 125 Val Tyr Asn Leu Leu Ser Leu Arg Phe Asn Ser Arg Ile Arg ValLys 130 135 140 Thr Tyr Thr Asp Glu Leu Thr Pro Ile Glu Ser Ala Val SerVal Phe 145 150 155 160 Lys Ala Ala Asn Trp Tyr Glu Arg Glu Ile Trp AspMet Phe Gly Val 165 170 175 Phe Phe Ala Asn His Pro Asp Leu Arg Arg IleLeu Thr Asp Tyr Gly 180 185 190 Phe Glu Gly His Pro Phe Arg Lys Asp PhePro Leu Ser Gly Tyr Val 195 200 205 Glu Leu Arg Tyr Asp Asp Glu Val LysArg Val Val Ala Glu Pro Val 210 215 220 Glu Leu Ala Gln Glu Phe Arg LysPhe Asp Leu Asn Ser Pro Trp Glu 225 230 235 240 Ala Phe Pro Val Tyr ArgGln Pro Pro Glu Ser Leu Lys Leu Glu Ala 245 250 255 Gly Asp Lys Lys ProAsp Ala Lys 260 1023 base pairs nucleic acid single linear ConsensusConsensus 2 GAACTCTAAT ACGAGCACTA TAGGGAAAGC TGGTAGCCTG CAGGTACCGGTCCGGAATTC 60 CCGGGTCGAC CCACGCGTCC GCCGTGCCCT TGGGGCTCCG TGTCCTGCTGTCTTTCCGTC 120 CGCTGCCTAG TCTGCATCTG AGTAACATGG CGGCGGCGGC GGTAGCCAGGCTGTGGTGGC 180 GCGGGATCTT GGGGGCCTCG GCGCTGACCA GGGGGACTGG GCGACCCTCCGTTCTGTTGC 240 TGCCGGTGAG GCGGGAGAGC GCCGGGGCCG ACACGCGCCC CACTGTCAGACCACGGAATG 300 ATGTGGCCCA CAAGCAGCTC TCAGCTTTTG GAGAGTATGT GGCTGAAATCTTGCCCAAGT 360 ATGTCCAACA AGTTCAGGTG TCCTGCTTCA ATGAGTTAGA GGTCTGTATCCATCCTGATG 420 GCGTCATCCC AGTGCTGACT TTCCTCAGGG ATCACACCAA TGCACAGTTCAAATCTCTGG 480 TTGACTTGAC AGCAGTGGAC GTCCCAACTC GGCAAAACCG TTTTGAGATTGTCTACAACC 540 TGTTGTCTCT GCGCTTCAAC TCACGGATCC GTGTGAAGAC CTACACAGATGAGCTGACGC 600 CCATTGAGTC TGCTGTCTCT GTGTTCAAGG CAGCCAACTG GTATGAAAGGGAGATCTGGG 660 ACATGTTTGG AGTCTTCTTT GCTAACCACC CTGATCTAAG AAGGATCCTGACAGATTATG 720 GCTTCGAGGG ACATCCTTTC CGGAAAGACT TTCCTCTATC TGGCTATGTTGAGTTACGTT 780 ATGATGATGA AGTGAAGCGT GTGGTGGCAG AGCCGGTGGA GTTGGCCCAAGAGTTCCGCA 840 AATTTGACCT GAACAGCCCC TGGGAGGCTT TCCCAGTCTA TCGCCAACCCCCGGAGAGTC 900 TCAAGCTTGA AGCCGGAGAC AAGAAGCCTG ATGCCAAGTA GCTCCAGGGAACGCATGTGG 960 ATCCTAGACA GCGCCTTATC TATGATTGAG TGTCCGTGTA AATAAATTCCTACTTAGACT 1020 TAC 1023 129 amino acids amino acid single linearConsensus Consensus 3 Met Ser Phe Pro Lys Tyr Lys Pro Ser Ser Leu ArgThr Leu Pro Glu 1 5 10 15 Thr Leu Asp Pro Ala Glu Tyr Asn Ile Ser ProGlu Thr Arg Arg Ala 20 25 30 Gln Ala Glu Arg Leu Ala Ile Arg Ala Gln LeuLys Arg Glu Tyr Leu 35 40 45 Leu Gln Tyr Asn Asp Pro Asn Arg Arg Gly LeuIle Glu Asn Pro Ala 50 55 60 Leu Leu Arg Trp Ala Tyr Ala Arg Thr Ile AsnVal Tyr Pro Asn Phe 65 70 75 80 Arg Pro Thr Pro Lys Asn Ser Leu Met GlyAla Leu Cys Gly Phe Gly 85 90 95 Pro Leu Ile Phe Ile Tyr Tyr Ile Ile LysThr Glu Arg Asp Arg Lys 100 105 110 Glu Lys Leu Ile Gln Glu Gly Lys LeuAsp Arg Thr Phe His Leu Ser 115 120 125 Tyr 451 base pairs nucleic acidsingle linear Consensus Consensus 4 CCAAGATGTC GTTCCCAAAG TATAAGCCGTCGAGCCTGCG CACTCTGCCT GAGACCCTCG 60 ACCCAGCCGA ATACAACATA TCTCCGGAAACCCGGCGGGC GCAAGCGGAG CGGTTGGCCA 120 TAAGAGCCCA GCTGAAACGA GAGTACCTGCTTCAGTACAA CGATCCCAAC CGCCGAGGGC 180 TCATCGAAAA TCCTGCCTTG CTTCGTTGGGCCTATGCAAG AACAATAAAT GTCTATCCTA 240 ATTTCAGACC CACTCCTAAA AACTCACTCATGGGAGCTCT GTGTGGATTT GGGCCCCTCA 300 TCTTCATTTA TTATATTATC AAAACTGAGAGGGATAGGAA AGAAAAACTT ATCCAGGAAG 360 GAAAATTGGA TCGAACATTT CACCTCTCATATTAAGTCTG GCAATGATGA CTATATGTAT 420 TCCTGCCTAA ATAAATCATC TATTAATCAT T451 106 amino acids amino acid single linear Consensus Consensus 5 MetPro Phe Leu Asp Ile Gln Lys Arg Phe Gly Leu Asn Ile Asp Arg 1 5 10 15Trp Leu Thr Ile Gln Ser Gly Glu Gln Pro Tyr Lys Met Ala Gly Arg 20 25 30Cys His Ala Phe Glu Lys Glu Trp Ile Glu Cys Ala His Gly Ile Gly 35 40 45Tyr Thr Arg Ala Glu Lys Glu Cys Lys Ile Glu Tyr Asp Asp Phe Val 50 55 60Glu Cys Leu Leu Arg Gln Lys Thr Met Arg Arg Ala Gly Thr Ile Arg 65 70 7580 Lys Gln Arg Asp Lys Leu Ile Lys Glu Gly Lys Tyr Thr Pro Pro Pro 85 9095 His His Ile Gly Lys Gly Glu Pro Arg Pro 100 105 470 base pairsnucleic acid single linear Consensus Consensus 6 AGCTAGTCGT TCTGAAGCGGCGGCCAGAGA AGAGTCAAGG GCACGAGCAT CGGCCATGCC 60 TTTCTTGGAC ATCCAGAAAAGGTTCGGCCT TAACATAGAT CGATGGTTGA CAATCCAGAG 120 TGGTGAACAG CCCTACAAGATGGCTGGTCG ATGCCATGCT TTTGAAAAAG AATGGATAGA 180 ATGTGCACAT GGAATCGGTTATACTCGGGC AGAGAAAGAG TGCAAGATAG AATATGATGA 240 TTTCGTAGAG TGTTTGCTTCGGCAGAAAAC GATGAGACGT GCAGGTACCA TCAGGAAGCA 300 GCGGGATAAG CTGATAAAGGAAGGAAAGTA CACCCCTCCA CCTCACCACA TTGGCAAGGG 360 GGAGCCTCGG CCCTGAACAGAGCAGCTGCT GATGTCTGGA GGCTGATTTT CCTGTTCTCT 420 GTTCTCCACT GGAAAGGTTGTTTACGACAA ACCTCCTTGT CAAAGTGTGT 470 119 amino acids amino acid singlelinear Consensus Consensus 7 Met Ile Ala Arg Arg Asn Pro Glu Pro Leu ArgPhe Leu Pro Asp Glu 1 5 10 15 Ala Arg Ser Leu Pro Pro Pro Lys Leu ThrAsp Pro Arg Leu Leu Tyr 20 25 30 Ile Gly Phe Leu Gly Tyr Cys Ser Gly LeuIle Asp Asn Leu Ile Arg 35 40 45 Arg Arg Pro Ile Ala Thr Ala Gly Leu HisArg Gln Xaa Xaa Tyr Ile 50 55 60 Thr Ala Phe Phe Phe Ala Gly Tyr Tyr XaaVal Lys Arg Glu Asp Tyr 65 70 75 80 Leu Tyr Ala Val Arg Asp Arg Glu MetPhe Gly Tyr Met Lys Leu His 85 90 95 Pro Glu Asp Phe Pro Glu Glu Asp LysLys Thr Tyr Gly Glu Ile Phe 100 105 110 Glu Lys Phe His Pro Ile Arg 115576 base pairs nucleic acid single linear Consensus Consensus 8CGCAGAGGAG GAGGAGAAAG CTGACCGCTT AGGCCCGGGT AGTGGTCGTC GTGGTTTTCC 60TTGTAGTTCG TGGTCTGAGA CCAGGCCTCA AGTGGAAACG GCGTCACCAT GATCGCACGG 120CGGAACCCAG AACCCTTACG GTTTCTGCCG GATGAGGCCC GGAGCCTGCC CCCGCCCAAG 180CTGACCGACC CGCGGCTCCT CTACATCGGC TTCTTGGGCT ACTGCTCCGG CCTGATTGAT 240AACCTGATCC GGCGGAGGCC GATCGCGACG GCTGGTTTGC ATCGCCAGNT TNTATATATT 300ACGGCCTTTT TTTTTGCTGG ATATTATNTT GTAAAACGTG AAGACTACCT GTATGCTGTG 360AGGGACCGTG AAATGTTTGG ATATATGAAA TTACATCCAG AGGATTTTCC TGAAGAAGAT 420AAGAAAACAT ATGGTGAAAT TTTTGAAAAA TTCCATCCAA TACGTTGAAG TCTTCAAAAT 480GCTTGCTCCA GTTTCACTGA TACCTGCTGT TTCTGAATTT GATGGAACAT GTTTCTTATG 540ACAGTTGAAG CTTATGCTAA TCTGTATGTT GACACC 576 266 amino acids amino acidsingle linear GenBank 163416 9 Met Ala Ala Ala Val Ala Ala Ala Ala ProGly Cys Trp Gln Arg Leu 1 5 10 15 Val Gly Ser Ala Ala Pro Ala Arg ValAla Gly Arg Pro Ser Val Leu 20 25 30 Leu Leu Pro Val Arg Arg Glu Ser SerAla Ala Asp Thr Arg Pro Thr 35 40 45 Val Arg Pro Arg Asn Asp Val Ala HisLys Gln Leu Ser Ala Phe Gly 50 55 60 Glu Tyr Val Ala Glu Ile Leu Pro LysTyr Val Gln Gln Val Gln Val 65 70 75 80 Ser Cys Phe Asn Glu Leu Glu IleCys Ile His Pro Asp Gly Val Ile 85 90 95 Pro Val Leu Thr Phe Leu Arg AspHis Ser Asn Ala Gln Phe Lys Ser 100 105 110 Leu Ala Asp Leu Thr Ala ValAsp Ile Pro Thr Arg Gln Asn Arg Phe 115 120 125 Glu Ile Val Tyr Asn LeuLeu Ser Leu Arg Phe Asn Ser Arg Ile Arg 130 135 140 Val Lys Thr Tyr ThrAsp Glu Leu Thr Pro Ile Glu Ser Ser Val Pro 145 150 155 160 Val Tyr LysAla Ala Asn Trp Tyr Glu Arg Glu Ile Trp Asp Met Phe 165 170 175 Gly ValPhe Phe Ala Asn His Pro Asp Leu Arg Arg Ile Leu Thr Asp 180 185 190 TyrGly Phe Glu Gly His Pro Phe Arg Lys Asp Phe Pro Leu Ser Gly 195 200 205Tyr Val Glu Leu Arg Tyr Asp Asp Glu Val Lys Arg Val Val Ala Glu 210 215220 Pro Val Glu Leu Ala Gln Glu Phe Arg Lys Phe Asp Leu Asn Ser Pro 225230 235 240 Trp Glu Ala Phe Pro Ala Tyr Arg Gln Pro Pro Glu Ser Leu LysLeu 245 250 255 Glu Ala Gly Asp Thr Lys Pro Glu Ala Lys 260 265 129amino acids amino acid single linear GenBank 114 10 Met Ser Phe Pro LysTyr Glu Ala Ser Arg Leu Ser Ser Leu Pro Thr 1 5 10 15 Thr Leu Asp ProAla Glu Tyr Asp Ile Ser Ser Glu Thr Arg Lys Ala 20 25 30 Gln Ala Glu ArgLeu Ala Ile Arg Ser Arg Leu Lys Arg Glu Tyr Gln 35 40 45 Leu Gln Tyr TyrAsp Pro Ser Arg Arg Gly Val Ile Glu Asp Pro Ala 50 55 60 Leu Val Arg TrpThr Tyr Ala Arg Ser Ala Asn Ile Tyr Pro Asn Phe 65 70 75 80 Arg Pro AsnThr Lys Thr Ser Leu Leu Gly Ala Leu Phe Gly Ile Gly 85 90 95 Pro Leu ValPhe Trp Tyr Tyr Val Phe Lys Thr Asp Arg Asp Arg Lys 100 105 110 Glu LysLeu Ile Gln Glu Gly Lys Leu Asp Arg Thr Phe Asn Ile Ser 115 120 125 Tyr106 amino acids amino acid single linear GenBank 224 11 Met Pro Phe PheAsp Val Gln Lys Arg Leu Gly Val Asp Leu Asp Arg 1 5 10 15 Trp Met ThrIle Gln Ser Ala Glu Gln Pro His Lys Ile Pro Ser Arg 20 25 30 Cys His AlaPhe Glu Lys Glu Trp Ile Glu Cys Ala His Gly Ile Gly 35 40 45 Ser Ile ArgAla Glu Lys Glu Cys Lys Ile Glu Phe Glu Asp Phe Arg 50 55 60 Glu Cys LeuLeu Arg Gln Lys Thr Met Lys Arg Leu His Ala Ile Arg 65 70 75 80 Arg GlnArg Glu Lys Leu Ile Lys Glu Gly Lys Tyr Thr Pro Pro Pro 85 90 95 His HisSer Gly Gln Glu Glu Pro Arg Ser 100 105 120 amino acids amino acidsingle linear GenBank 582 12 Met Met Thr Gly Arg Gln Gly Arg Ala Thr PheGln Phe Leu Pro Asp 1 5 10 15 Glu Ala Arg Ser Leu Pro Pro Pro Lys LeuThr Asp Pro Arg Leu Ala 20 25 30 Phe Val Gly Phe Leu Gly Tyr Cys Ser GlyLeu Ile Asp Asn Ala Ile 35 40 45 Arg Arg Arg Pro Val Leu Leu Ala Gly LeuHis Arg Gln Leu Leu Tyr 50 55 60 Ile Thr Ser Phe Val Phe Val Gly Tyr TyrLeu Leu Lys Arg Gln Asp 65 70 75 80 Tyr Met Tyr Ala Val Arg Asp His AspMet Phe Ser Tyr Ile Lys Ser 85 90 95 His Pro Glu Asp Phe Pro Glu Lys AspLys Lys Thr Tyr Gly Glu Val 100 105 110 Phe Glu Glu Phe His Pro Val Arg115 120

What is claimed is:
 1. An isolated polypeptide selected from the groupconsisting of: a) a recombinant human polypeptide comprising the aminoacid sequence of SEQ ID NO:1, said recombinant polypeptide being free ofother human amino acid sequences, and b) a recombinant polypeptidecomprising a naturally occurring human amino acid sequence at least 90%identical to the amino acid sequence of SEQ ID NO:1, said recombinantpolypeptide being free of other human amino acid sequences.
 2. Anisolated polypeptide of claim 1, comprising the amino acid sequence ofSEQ ID NO:1.
 3. An isolated polypeptide of claim 1, comprising anaturally occurring human amino acid sequence at least 90% identical tothe amino acid sequence of SEQ ID NO:1, said polypeptide being free ofother human amino acid sequences.
 4. An isolated polypeptide of claim 1,comprising a naturally occurring human amino acid sequence at least 95%identical to the amino acid sequence of SEQ ID NO:1, said polypeptidebeing free of other human amino acid sequences.
 5. A compositioncomprising a polypeptide of claim 1 and a pharmaceutically acceptableexcipient.
 6. A composition comprising a polypeptide of claim 2 and apharmaceutically acceptable excipient.
 7. A composition comprising apolypeptide of claim 3 and a pharmaceutically acceptable excipient.
 8. Acomposition comprising a polypeptide of claim 4 and a pharmaceuticallyacceptable excipient.
 9. A method for producing a polypeptide of claim1, the method comprising: a) culturing a cell under conditions suitablefor expression of the polypeptide, wherein said cell is transformed witha recombinant polynucleotide, and said recombinant polynucleotidecomprises a promoter sequence operably linked to a polynucleotideencoding the polypeptide of claim 1, and b) recovering the polypeptideso expressed.
 10. A method for screening a compound for effectiveness asan agonist of a polypeptide of claim 1, the method comprising: a)exposing a sample comprising a polypeptide of claim 1 to a compound, andb) detecting agonist activity in the sample.
 11. A method for screeninga compound for effectiveness as an antagonist of a polypeptide of claim1, the method comprising: a) exposing a sample comprising a polypeptideof claim 1 to a compound, and b) detecting antagonist activity in thesample.
 12. A method of screening for a compound that specifically bindsto the polypeptide of claim 1, said method comprising the steps of: a)combining the polypeptide of claim 1 with at least one test compoundunder suitable conditions, and b) detecting binding of the polypeptideof claim 1 to the test compound, thereby identifying a compound thatspecifically binds to the polypeptide of claim
 1. 13. A method ofscreening for a compound that modulates the activity of the polypeptideof claim 1, said method comprising: a) combining the polypeptide ofclaim 1 with at least one test compound under conditions permissive forthe activity of the polypeptide of claim 1, b) assessing the activity ofthe polypeptide of claim 1 in the presence of the test compound, and c)comparing the activity of the polypeptide of claim 1 in the presence ofthe test compound with the activity of the polypeptide of claim 1 in theabsence of the test compound, wherein a change in the activity of thepolypeptide of claim 1 in the presence of the test compound isindicative of a compound that modulates the activity of the polypeptideof claim 1.